Methods for preparing therapeutically active cells using microfluidics

ABSTRACT

The present invention is directed to the use of microfluidics in the preparation of cells and compositions for therapeutic uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/553,723, filed on Sep. 1, 2017; the benefit of U.S.Provisional Patent Application No. 62/567,553, filed on Oct. 3, 2017;the benefit of Provisional Patent Application No. 62/635,304, filed onFeb. 26, 2018; and the benefit of Provisional Patent Application No.62/656,939, filed on Apr. 12, 2018; and, in addition, the application isa continuation-in-part of PCT/US2017/057876, filed on Oct. 23, 2017.These prior applications are all incorporated by reference herein intheir entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. CA174121and No. HL110574 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed primarily to methods of preparingcells and compositions for therapeutic uses. The methods employmicrofluidic devices that separate cells based on size.

BACKGROUND OF THE INVENTION

Cell therapy, and especially CAR-T cell therapy, has demonstratedextraordinary efficacy in treating B-cell diseases such as B-acutelymphoid leukemia (B-ALL) and B-Cell Lymphomas. As a result, the demandfor autologous therapies has increased dramatically and developmentefforts have broadened to focus on cancers characterized by solidtumors, such as glioblastomas (Vonderheide, et al., Immunol. Rev.257:7-13 (2014); Fousek, et al., Clin. Cancer Res. 21:3384-3392 (2015);Wang, et al., Mol. Ther. Oncolytics 3:16015 (2016); Sadelain, et al.,Nature 545:423-431 (2017)). Targeted gene editing with CRISPR/Cas-9 infocused populations of autologous cells, such as stem cells, may furtherfuel demand (Johnson, et al., Cancer Cell Res. 27:38-58 (2017)).

The preparation of cells for personalized therapy is usually alabor-intensive process that relies on procedures adapted from bloodbanking or protein bioprocessing procedures which are poorly suited fortherapeutic applications. Cell losses associated with processing stepsare typically substantial (Hokland, et al., Scand. J. Immunol.11:353-356 (1980); Stroncek, et al., J. Transl. Med. 12:241 (2014)), inpart because of processes that use preparations that achieve cellspecific separations (Powell, et al., Cytotherapy 11:923-935 (2009);TerumoBCT. ELUTRA Cell Separation System. Manufacturer recommendationsfor the Enrichment of Lymphocytes from Apheresis Residues) but do so atthe expense of cell viability and yield (Chiche-Lapierre, Cytotherapy18(6):S47 (2016)). Thus, there is a need for more efficient processes.

SUMMARY OF THE INVENTION

The present invention is directed, inter alia, to methods of collectingand rapidly processing cells, particularly cells that have therapeuticuses. Many of the methods rely on Deterministic Lateral Displacement(DLD), a process that involves flowing a sample through a microfluidicdevice containing a specifically designed array of microposts that aretilted at a small angle from the direction of fluid flow (Davis, et al.,Proc. Natl. Acad. Sci. USA 103:14779-14784 (2006); Inglis, et al., LabChip 6:655-658 (2006); Chen, et al., Biomicrofluidics. 9(5):054105(2015)). Cells larger than the target size of the micropost array may begently deflected (“bumped”) by the microposts into a stream of cleanbuffer, effectively separating them from smaller, non-deflected cellsand particles, while simultaneously washing the cells in a process thatis non-injurious. Advantageous characteristics of DLD with respect tocell processing are described in Table 1:

TABLE 1 Intrinsic Properties of DLD and Their Implications for CellProcessing DLD Feature Enablement Implications Uniform Fractionatecomplex Uniform and gentle de-bulking of platelet and feature andmixtures based on size with RBC from blood products without gap sizeability to discriminate centrifugation up to 99.99% efficiency particlesto within ~0.5 μm. Eliminates open solutions such as Ficoll, and avoidsneed for harsh hypertonic solutions (Elutriation). Ability to mixdifferent Dc Use of sequential cut-offs to manage highly within the samedevice heterogeneous fractionations Cell Washing & Buffer CellWashing >99.9% removal in single pass Exchange Potential to improve andremove cell culture while maintaining closed system ensuring viablecells. Concentration Concentration of cells in culture to makedownstream processing seamless. Minimize reagent expense withoutrequiring open centrifugation or transfer losses. Closeable Simple,sterilizable Ideal for single use, especially patient specific fluidpath therapeutic device. Low Dead <50 μl Dead volume per 14 Excellentcell recovery Volume lane chip Requires only Hands free operationPotential to automate complex cell handling positive and liquid additionexchange processes within a pressure closed system

Methods for Engineering Target Cells

In its first aspect, the invention is directed to a method ofgenetically engineering a population of target cells. This is done byisolating the target cells from a crude fluid composition by performingDeterministic Lateral Displacement (DLD) on a microfluidic device. Thedevice is characterized by the presence of at least one channel whichextends from a sample inlet to one or more fluid outlets, and which isbounded by a first wall and a second wall opposite from the first wall.An array of obstacles is arranged in rows in the channel, with eachsubsequent row of obstacles being shifted laterally with respect to aprevious row. The obstacles are disposed in a manner such that, when thecrude fluid composition is applied to an inlet of the device and passedthrough the channel, target cells flow to one or more collection outletswhere an enriched product is collected, and contaminant cells orparticles flow to one or more waste outlets that are separate from thecollection outlets. Once the target cells have been purified using thedevice, they are transfected or transduced with nucleic acids designedto impart upon the cells a desired phenotype, e.g., to express achimeric molecule (preferably a protein that makes the cells oftherapeutic value). The population of cells may then be expanded byculturing in vitro. When cultured and expanded, the yield ofrecombinantly engineered target cells exhibiting the desired phenotypeis preferably at least 10% greater than identical cells not subjected toDLD (and particularly cells that have been exposed to Ficollcentrifugation but not DLD), and more preferably at least 20, 30, 40, or50% greater.

In a preferred embodiment, the crude fluid composition is blood or, morepreferably, a preparation of leukocytes that has been obtained byperforming apheresis or leukapheresis on the blood of a patient.Preferred target cells include T cells, B-cells, NK-cells, monocytes andprogenitor cells, with T cells (especially natural killer T cells) beingthe most preferred. Apart from leukocytes, other types of cells, e.g.,dendritic cells or stem cells, may also serve as target cells.

In general, crude fluid compositions containing target cells will beprocessed without freezing (at least up until the time that they aregenetically engineered), and at the site of collection. The crude fluidcomposition will preferably be the blood of a patient, and morepreferably be a composition containing leukocytes obtained as the resultof performing apheresis or leukapheresis on such blood. However, theterm “crude fluid composition” also includes bodily fluids such as lymphor synovial fluid as well as fluid compositions prepared from bonemarrow or other tissues. The crude fluid composition may also be derivedfrom tumors or other abnormal tissue.

Although it is not essential that target cells be bound to a carrierbefore being genetically engineered, it is preferred that, either beforeor after DLD is first performed (preferably before) they be bound to oneor more carriers. The exact means by which this occurs is not criticalto the invention but binding should be done “in a way that promotes DLDseparation.” This term, as used in the present context, means that themethod must ultimately result in binding that exhibits specificity for aparticular target cell type, that provides for an increase in size ofthe complex relative to the unbound cell of at least 2 μm (andalternatively at least 20, 50, 100, 200, 500 or 1000% when expressed asa percentage) and, in cases where therapeutic or other uses require freetarget cells, that allow the target cell to be released from complexesby chemical or enzymatic cleavage, chemical dissolution, digestion, dueto competition with other binders, by physical shearing, e.g., using apipette to create shear stress, or by other means.

In a preferred embodiment, the carriers have on their surface anaffinity agent (e.g., an antibody, activator, hapten, aptamer, nucleicacid sequence, or other compound) that allows the carriers to binddirectly to the target cells with specificity. Alternatively, there maybe an intermediary protein, cell, or other agent that binds to both thetarget cell and carrier with specificity. For example, antibodies may beused that recognize surface antigens on target cells and that also bindwith specificity to carriers (e.g., due to that presence of a secondantibody on the carrier surface, avidin/biotin binding or some othersimilar interaction). In addition, target cells may sometimes interactwith specificity with other cells to form a complex and in so doing, theother cells may serve as a biological carrier, i.e., they may increasethe effective size of the target cell and thereby facilitate itsseparation from uncomplexed cells. For example, human T cells mayinteract with sheep erythrocytes or autologous human erythrocytes toform a rosette of cells that can then be purified as a complex.Alternatively, other carriers may bind with specificity to cells in sucha rosette to further promote a size based separation.

As used in this context, the word “specificity” means that at least 100(and preferably at least 1000) target cells will be bound by carrier inthe crude fluid composition relative to each non-target cell bound. Incases where the carrier binds after DLD, the binding may occur eitherbefore the target cells are genetically engineered or after.

Binding of the carriers may help to stabilize cells, activate them(e.g., to divide) or help to facilitate the isolation of one type ofcell from another. As suggested above, the binding of carriers to cellscan take place at various times in the method, including during the timethat cells are being obtained. In order to improve separation, carriersmay be chosen such that the binding of a single carrier to a cellresults in a carrier-cell complex that is substantially larger than thesize of the cell alone. Alternatively carriers may be used that aresmaller that the target cell. In this case, it is preferred that severalcarriers bind with specificity to a cell, thereby forming a complexhaving one cell and multiple carriers. During DLD, complexed targetcells may separate from uncomplexed cells having a similar size andprovide a purification that would otherwise not occur.

In order to achieve such separation, the diameter of the complex shouldpreferably be at least 20% larger than the uncomplexed target cells andmore preferably at least 50% larger, at least twice as large or at leastten times as large. As stated above this increase in size may be eitherdue to the binding of a single large carrier to target cells or due tothe binding of several smaller carriers. This may be accomplished using:a) only carriers with a diameter at least as large (or in otherembodiments, at least twice as large or at least ten times as large) asthat of the target cells; b) only carriers with a diameter no more than50% (or in other embodiments, no more than 25% or 15%) as large as thatof the target cells; or c) mixtures of large and small carriers withthese size characteristics (e.g., there may be one group of carrierswith a diameter at least as large (or at least twice or ten times aslarge) as the target cells and a second group of carriers with adiameter no more than 50% (or no more than 25% or 15%) as large as thatof the target cells. Typically, a carrier will have a diameter of 1-1000μm (and often in the range of 5-600 or 5-400 μm). Ideally, the complexeswill be separated from other cells or contaminants by DLD on amicrofluidic device having an array of obstacles with a critical sizelower than the size of the complexes but higher than the size ofuncomplexed non-target cells or contaminants.

In addition carriers may act in a way that “complements DLD separation”rather than directly promoting separation by this technique. Forexample, a carrier (e.g., as Janus or Strawberry-like particles) maycomprise two or more discrete chemical properties that support andconfer actionable differential non-size related secondary properties,such as chemical, electrochemical, or magnetic properties, on the cellsthat they bind with and these properties may be used in downstreamprocesses. Thus, the particles may be used to facilitate magneticseparation, electroporation, or gene transfer. They may also conferadvantageous changes in cellular properties relating to, for example,metabolism or reproduction.

In a particularly important embodiment, the binding of carriers may beused as a means of separating a specific leukocyte, especially T cells,including natural killer T cells, from other leukocytes, e.g.,granulocytes and monocytes, and/or from other cells. This may be done,for example, in a two step process in which DLD is performed on targetcells that are not bound to a carrier using an array of obstacles with acritical size smaller than the cells and also performed on complexescomprising target cells and carriers using an array of obstacles with acritical size smaller than the complexes but larger than the uncomplexedcells. The DLD steps can be performed in either order, i.e., DLD may beperformed on the complexes before or after being performed onuncomplexed target cells.

No more than four hours (and preferably no more than three, two or onehour(s)) should elapse from the time that the obtaining of crude fluidcomposition is completed until the target cells are first bound tocarriers. In addition, no more that five hours (and preferably no morethan four, three or two hours) should elapse from the time that theobtaining of crude fluid composition is completed until the first timethat target cells are transfected or transduced.

In a particularly preferred embodiment, the target cells in the methodsdescribed above are T cells (especially natural killer T cells andmemory T cells) and these are engineered to express chimeric antigenreceptors on their surface. The procedures for making these CAR T cellsare described more specifically below.

Methods for Making CAR T Cells

The invention includes a method of producing CAR T cells by obtaining acrude fluid composition comprising T cells (especially natural killer Tcells and memory T cells) and performing DLD on the composition using amicrofluidic device. Generally, the crude fluid composition comprising Tcells will be an apheresis or leukapheresis product derived from theblood of a patient and containing leukocytes.

The microfluidic device must have at least one channel extending from asample inlet to one or more fluid outlets, wherein the channel isbounded by a first wall and a second wall opposite from the first wall.An array of obstacles is arranged in rows in the channel, eachsubsequent row of obstacles being shifted laterally with respect to aprevious row. These obstacles are disposed in a manner such that, whenthe crude fluid composition comprising T cells is applied to an inlet ofthe device and fluidically passed through the channel, the T cells flowto one or more collection outlets where an enriched product is collectedand other cells (e.g., red blood cells, and platelets) or otherparticles of a different (generally smaller) size than the T cells flowto one or more waste outlets that are separate from the collectionoutlets. Once obtained, the T cells are genetically engineered toproduce chimeric antigen receptors (CARs) on their surface usingprocedures well established in the art. These receptors should generallybind antigens that are on the surface of a cell associated with adisease or abnormal condition. For example, the receptors may bindantigens that are unique to, or overexpressed on, the surface of cancercells. In this regard, CD19 may sometimes be such an antigen.

The genetic engineering of CAR-expressing T cells will generallycomprise transfecting or transducing T cells with nucleic acids and,once produced, the CAR T cells may be expanded in number by growing thecells in vitro. Activators or other factors may be added during thisprocess to promote growth, with IL-2 and IL-15 being among the agentsthat may be used. The yield of T cells expressing chimeric receptors ontheir surface after DLD, recombinant engineering and expansion, should,in some embodiments be at least 10% greater than T cells prepared in thesame manner but not subjected to DLD and preferably at least 20, 30, 40or 50% greater. Similarly, in some embodiments, the yield of T cellsexpressing the chimeric receptors on their surface should be at least10% greater than T cells isolated by Ficoll centrifugation and notsubjected to DLD and preferably at least 20, 30, 40 or 50% greater.

Chimeric receptors will typically have a) an extracellular region withan antigen binding domain; b) a transmembrane region and c) anintracellular region. The cells may also be recombinantly engineeredwith sequences that provide the cells with a molecular switch that, whentriggered, reduce CAR T cell number or activity. In a preferredembodiment, the antigen binding domain is a single chain variablefragment (scFv) from the antigen binding regions of both heavy and lightchains of a monoclonal antibody. There is also preferably a hinge regionof 2-20 amino acids connecting the extracellular region and thetransmembrane region. The transmembrane region may have CD3 zeta, CD4,CD8, or CD28 protein sequences and the intracellular region should havea signaling domain, typically derived from CD3-zeta, CD137 or a CD28.Other signaling sequences may also be included that serve to regulate orstimulate activity.

After obtaining the crude fluid composition comprising T cells, orduring the time that they are being collected, the T cells may, for thereasons discussed above, be bound to one or more carriers in a way thatpromotes DLD separation. This will preferably take place beforeperforming DLD. However, it may also occur after performing DLD andeither before or after cells are transfected or transduced for the firsttime. In a preferred embodiment, the carriers should comprise on theirsurface an affinity agent (e.g., an antibody, activator, hapten oraptamer) that binds with specificity to T cells, preferably naturalkiller T cells. The term “specificity” as used in this context meansthat the carriers bind preferentially to the desired T cells as comparedto any other cells in the composition. For example, the carriers maybind to 100 or 1000 CD8+ T cells for each instance in which it binds adifferent type of cell.

Carriers may, in some embodiments, have a spherical shape and be made ofeither biological or synthetic material, including collagen,polysaccharides including polystyrene, acrylamide, alginate and magneticmaterial. In addition, carriers may act in a way that complements DLDseparation.

In order to aid in achieving a separation, the diameter of the complexformed between T cells and carriers should preferably be at least 20%larger than the uncomplexed T cells and preferably at least 50% larger,at least twice as large or at least ten times as large. This increase insize may be either due to the binding of a single large carrier to thecells or due to the binding of several smaller carriers. Binding mayinvolve using: a) only carriers with a diameter at least as large (or inother embodiments, at least twice as large or at least ten times aslarge) as that of the T cells; b) only carriers with a diameter no morethan 50% (or in other embodiments, no more than 25% or 15%) as large asthat of the T cells; or c) mixtures of large and small carriers withthese size characteristics (e.g., there may be one group of carrierswith a diameter at least as large (or at least twice or ten times aslarge) as the T cells and a second group of carriers with a diameter nomore than 50% (or no more than 25% or 15%) as large as that of the Tcells. Typically a carrier will have a diameter of 1-1000 μm (and oftenin the range of 5-600 or 5-400 μm). Ideally, the complexes will beseparated from uncomplexed cells or contaminants by DLD on amicrofluidic device having an array of obstacles with a critical sizelower than the size of the complexes but higher than the size ofuncomplexed non-target cells or contaminants.

As discussed above in connection with target cells, the purification ofT cells may involve a two step process. For example, DLD may beperformed on T cells that are not bound to carriers using an array ofobstacles with a critical size smaller than the T cells. A compositioncontaining the separated T cells together with other cells or particlesmay then be recovered and bound to one or more carriers in a way thatpromotes DLD separation and in which T cells are bound with specificity.The complexes thereby formed may then be separated on an array ofobstacles with a critical size smaller than the complexes but largerthan uncomplexed cells. In principle, the DLD steps could be performedin either order, i.e., it might be performed on the complexes first oron the uncomplexed T cells first.

Preferably, no more than four hours (and, more preferably, no more thanthree, two or one hour(s)) should elapse from the time that theobtaining of the crude fluid composition comprising T cells is completed(e.g., from the time that apheresis or leukapheresis is completed) untilthe T cells are bound to a carrier. In addition, no more than five hours(and preferably no more than four hours, three or two hours) shouldelapse from the time that the obtaining of T cells is completed untilthe first time that T cells are transfected or transduced. Ideally, allsteps in producing the CAR T cells are performed at the same facilitywhere the crude fluid composition comprising T cells is obtained and allsteps are completed in no more than four (and preferably no more thanthree) hours and without the cells being frozen.

Treating Cancer, Autoimmune Disease or Infectious Disease Using CAR TCells

In another aspect, the invention is directed to a method of treating apatient for cancer, an autoimmune disease or an infectious disease byadministering CAR T cells engineered to express chimeric antigenreceptors recognizing cancer cell antigens, or antigens on cellsresponsible for, or contributing to, autoimmune or infectious disease.The CAR T cells may be made using the methods discussed in the sectionabove, i.e., by obtaining a crude fluid composition comprising T cells(preferably a leukocyte-containing apheresis or leukapheresis productderived from the patient) and then performing DLD on the compositionusing a microfluidic device. The CAR T cells (preferably natural killerT cells, and memory T cells) recovered in this manner are then expandedby growing the cells in vitro. Finally, the cells are administered to apatient, which should generally be the same patient that gave the bloodfrom which the T cells were isolated.

Preferably, the yield of T cells expressing chimeric receptors on theirsurface after DLD, recombinant engineering and expansion is at least 10%greater than T cells prepared in the same manner but not subjected toDLD and more preferably at least 20, 30, 40 or 50% greater. For example,the yield of T cells expressing the chimeric receptors on their surfacemay be at least 10% greater than T cells isolated by Ficollcentrifugation and not subjected to DLD and preferably at least 20, 30,40 or 50% greater.

Chimeric receptors will typically have at least: a) an extracellularregion with an antigen binding domain; b) a transmembrane region and c)an intracellular region. The cells may also be recombinantly engineeredwith sequences that provide the cells with a molecular switch that, whentriggered, reduce CAR T cell number or activity. In a preferredembodiment, the antigen binding domain is a single chain variablefragment (scFv) from the antigen binding regions of both heavy and lightchains of a monoclonal antibody. There is also preferably a hinge regionof 2-20 amino acids connecting the extracellular region and thetransmembrane region. The transmembrane region itself may have CD3 zeta,CD4, CD8, or CD28 protein sequences and the intracellular region willhave a signaling domain, typically derived from CD3-zeta and/or a CD28intracellular domain. Other signaling sequences may also be includedthat serve to regulate or stimulate activity.

After obtaining the crude fluid composition or during the time the crudefluid composition is being collected, T cells present in the compositionmay be bound to one or more carriers in a way that promotes orcomplements DLD separation. This will preferably take place beforeperforming DLD. However, it may also occur after performing DLD andeither before or after the cells are genetically engineered. Preferablythe binding will promote DLD separation and the carriers will compriseon their surface an antibody, activator or other agent that binds withspecificity to T cells, especially natural killer T cells. The term“specificity” as used in this context means that the carrier will bebound preferentially to the desired T cells as compared to any othercells in the composition. For example, the carrier may bind to 100 or1000 CD8+ T cells for every carrier that binds to other types of cells.

The diameter of the complex formed between T cells and carrier shouldpreferably be at least 20% larger than the uncomplexed T cells and morepreferably at least 50% larger, at least twice as large or at least tentimes as large. This increase in size may be either due to the bindingof a single large carrier to the cells or due to the binding of severalsmaller carriers. Binding may involve using: a) only carriers with adiameter at least as large (or in other embodiments, at least twice aslarge or at least ten times as large) as that of the T cells; b) onlycarriers with a diameter no more than 50% (or in other embodiments, nomore than 25% or 15%) as large as that of the T cells; or c) mixtures oflarge and small carriers with these size characteristics (e.g., theremay be one group of carriers with a diameter at least as large (or atleast twice or ten times as large) as the T cells and a second group ofcarriers with a diameter no more than 50% (or no more than 25% or 15%)as large as that of the T cells. Typically, a carrier will have adiameter of 1-1000 μm (and often in the range of 5-600 or 5-400 μm).Ideally, the complexes will be separated from uncomplexed cells orcontaminants by DLD on a microfluidic device having an array ofobstacles with a critical size lower than the size of the complexes buthigher than the size of uncomplexed non-target cells or contaminants.

The purification of T cells may involve a two step process. For example,DLD may be performed on T cells that are not bound to carriers using anarray of obstacles with a critical size smaller than the T cells. Acomposition containing the separated T cells together with other cellsor particles may then be recovered and bound to one or more carriers ina way that promotes DLD separation and in which T cells are bound withspecificity. The complexes thereby formed may then be separated on anarray of obstacles with a critical size smaller than the complexes butlarger than uncomplexed cells. In principle, the DLD steps could beperformed in either order, i.e., it might be performed on the complexesfirst or on the uncomplexed T cells first.

Preferably, no more than four hours (and more preferably no more thanthree, two or one hour(s)) should elapse from the time that theobtaining of T cells is completed (e.g., until apheresis orleukapheresis is completed) until the T cells are bound to a carrier. Inaddition, no more than five hours (and preferably no more than four,three or two hours) should elapse from the time that the obtaining of Tcells is completed until the first time that T cells are transfected ortransduced. Ideally, all steps in producing the CAR T cells areperformed at the same facility where the crude fluid compositioncomprising T cells is obtained and all steps are completed in no morethan four (and preferably no more than three) hours.

CAR T cells made in this way may be used in treating patients forleukemia, e.g., acute lymphoblastic leukemia using procedures wellestablished in the art of clinical medicine and, in these cases, the CARmay recognize CD19 or CD20 as a tumor antigen. The method may also beused for solid tumors, in which case antigens recognized may includeCD22; RORI; mesothelin; CD33/IL3Ra; c-Met; PSMA; Glycolipid F77;EGFRvIII; GD-2; NY-ESO-1; MAGE A3; and combinations thereof with respectto autoimmune diseases, CAR T cells may be used to treat rheumatoidarthritis, lupus, multiple sclerosis, ankylosing spondylitis, type 1diabetes or vasculitis.

In some embodiments, the target cells produced by the methods describedabove will be available for administration to a patient earlier than ifthe cells were generated using methods not including a DLD. These cellsmay be administered 1 or more days earlier, and preferably 2, 3, 4, 5 ormore days earlier. The cells may be administered within 8-10 days fromthe time that obtaining of the crude fluid composition is completed.

Collection and Processing of Cells

The current invention is also directed to protocols for collecting andprocessing cells from a patient which are designed to process cellsquickly, and which can generally be performed at sites where the cellsare collected. The protocols may be used as a part of the methods forpreparing target cells and CAR T cells described above. Aspects of someof these protocols are illustrated in FIGS. 13 and 14 and may becontrasted with the protocol shown in FIG. 12. In the particularprocedures illustrated, a composition obtained by apheresis of wholeblood is obtained and T cells in the composition are then selected. Theterm “selected” in this context means that the T cells are bound byagents that recognize the T cells with specificity (as defined above).DLD is then used to isolate the selected T cells and transfer thesecells into a chosen fluid medium.

More generally, the invention concerns a method of collecting targetcells by: a) obtaining a crude fluid composition comprising the targetcells from a patient; and b) performing Deterministic LateralDisplacement (DLD) on the crude fluid composition to obtain acomposition enriched in target cells wherein either before, or afterDLD, the target cells are bound to a carrier in a way that promotes DLDseparation. For example, a carrier may be used that has on its surfacean affinity agent (e.g., an antibody, activator, hapten or aptamer) thatbinds with specificity (as defined above) to the target cells.

Carrier may, if desired, be bound to target cells during the time thatthe cells are being collected from the patient and no more than fivehours (and preferably no more than four, three, two or one hour(s))should elapse from the time that the obtaining of the crude fluidcomposition comprising target cells is completed until the target cellsare bound to the carrier.

The diameter of the complex formed between target cells and one or morecarriers should preferably be at least 20% larger than the uncomplexedcells and preferably at least 50% larger, at least twice as large or atleast ten times as large. This increase in size may be either due to thebinding of a single large carrier to the target cells or due to thebinding of several smaller carriers. Binding may involve using: i) onlycarriers with a diameter at least as large (or in other embodiments, atleast twice as large or at least ten times as large) as that of thetarget cells; ii) only carriers with a diameter no more than 50% (or inother embodiments, no more than 25% or 15%) as large as that of thetarget cells; or iii) mixtures of large and small carriers with thesesize characteristics (e.g., there may be one group of carriers with adiameter at least as large (or at least twice or ten times as large) asthe target cells and a second group of carriers with a diameter no morethan 50% (or no more than 25% or 15%) as large as that of the targetcells. Typically a carrier will have a diameter of 1-1000 μm (and oftenin the range of 5-600 or 5-400 μm). Ideally the complexes would beseparated from other cells or contaminants by DLD on a microfluidicdevice having an array of obstacles with a critical size lower than thesize of the complexes but higher than the size of uncomplexed cells orcontaminants.

In a preferred embodiment, the crude fluid composition comprising targetcells is obtained by performing apheresis or leukapheresis on blood fromthe patient. This composition may include one or more additives that actas anticoagulants or that prevent the activation of platelets. Examplesof such additives include ticlopidine, inosine, protocatechuic acid,acetylsalicylic acid, and tirofiban alone or in combination.

The microfluidic devices must have at least one channel extending from asample inlet to one or more fluid outlets, wherein the channel isbounded by a first wall and a second wall opposite from the first wall.There must also be an array of obstacles arranged in rows in thechannel, with each subsequent row of obstacles being shifted laterallywith respect to a previous row such that, when said crude fluidcomposition comprising target cells is applied to an inlet of the deviceand fluidically passed through the channel, target cells flow to one ormore collection outlets where an enriched product is collected andcontaminant cells, or particles that are in the crude fluid compositionand that are of a different size than the target cells flow to one morewaste outlets that are separate from the collection outlets.

In a particularly preferred embodiment, target cells are T cellsselected from the group consisting of: Natural Killer T cells; CentralMemory T cells; Helper T cells and Regulatory T cells, with NaturalKiller T cells being the most preferred. In alternative preferredembodiments, the target cells are stem cells, B cells, macrophages,monocytes, dendritic cells, or progenitor cells.

In addition to steps a) and b), the method of the invention may include:c) genetically engineering cells by transducing them using a viralvector. Alternatively, the cells may be transfected electrically,chemically or by means of nanoparticles and/or expanded cells in number;and/or d) treating the same patient from which the target cells wereobtained with the target cells collected. In addition, the collectedcells may be cultured and/or cryopreserved. In cases where the targetcells are T cells, culturing should generally be carried out in thepresence of an activator, preferably an activator that is bound to acarrier. Among the factors that may be included in T cell cultures areIL-2 and IL-15.

In some embodiments, the target cells produced by the methods describedabove will be available for administration to a patient earlier than ifthe cells were generated using methods not including DLD. These cellsmay be administered 1 or more days earlier, and preferably 2, 3, 4, 5 ormore days earlier. The cells may be administered within 8-10 days fromthe time that obtaining of the crude fluid composition is completed.

In addition to the methods discussed above, the invention includes thetarget cells produced by the methods and treatment methods in which thetarget cells are administered to a patient.

Altering the Characteristics of Leukocytes Using DLD

Reducing the level of platelets in leukocyte preparations has advantagesboth with respect to the making of CAR T cells and in preparingleukocytes for other therapeutic uses. In this regard, the presentinvention is based, in part, on the concept that DLD reduces the totalnumber of platelets in apheresis samples more effectively than commonlyused Ficoll separations (see FIGS. 19-21), especially when a buffer isused that does not promote platelet aggregation (see FIGS. 22-23). Whenused in combination with separation based on magnetic beads thatspecifically bind to T cells, DLD results in a preparation of cells thatcan be expanded more rapidly than when such magnetic beads are usedeither alone or in conjunction with Ficoll centrifugation (see FIG. 24).This effect may be partly due to a reduction in platelet number andpartly due to factors that are independent of the number of plateletspresent (see FIG. 24). In addition, the results obtained using theDLD/magnetic bead procedure are more consistent (see FIG. 25) and theexpanded T cells from this procedure have a higher percentage of T cellswith a central memory phenotype when compared to populations preparedusing a Ficoll/magnetic bead approach (see FIG. 26). The higher initialcell recovery from DLD combined with: a) a more rapid expansion of Tcells and b) a higher percentage of central memory cells, means thattherapeutically effective levels of T cells can be made available forpatients more rapidly.

In one aspect, the invention is directed to a method for decreasing theratio of platelets to leukocytes in an apheresis sample by performingdeterministic lateral displacement on the sample in the absence ofcentrifugation or elutriation to obtain a product in which the ratio ofplatelets to leukocytes is at least 20% (and preferably 50% or 70%)lower than the ratio obtained when the same procedure is performed usingcentrifugation (including gradient centrifugation or counterflowcentrifugation) or elutriation instead of DLD. Preferably, thisprocedure includes no separation steps performed on the apheresis sampleprior to DLD and DLD is carried out in a buffer that does not compriseintercalators or other means that alter the size of platelets and thatdoes not promote platelet aggregation. Agents that should be avoidedinclude dextran and other highly charge polymers. In addition tolowering the ratio of platelets to leukocytes, the total number ofplatelets in the DLD derived product should be at least 70% lower thanin the apheresis sample and preferably, at least 90% lower.

In another aspect, the invention is directed to a method for purifying Tcells from an apheresis sample by performing DLD on the sample, followedby an affinity separation step and expansion of the T cells by culturingin the presence of activator. This process should result in a number ofT cells that is at least twice as high as the number produced by thesame procedure performed using Ficoll centrifugation instead of DLD. Apreferred affinity method comprises the use of magnetic beads that bindspecifically to T cells by an antibody that recognizes at least CD3, andmight include CD3 together with CD28 or other costimulatory molecules.The number of T cells obtained after 14 days in culture should be atleast two times higher (and preferably at least four or six time higher)than the number produced by the same procedure performed using Ficollcentrifugation instead of DLD. The percentage of memory T cells in theproduct produced by this method should be at least 10% (and preferablyat least 20%) higher than the percentage produced using the sameprocedure but with Ficoll centrifugation instead of DLD.

The method is especially well suited to the production of T cells forCAR T cell therapy. The time needed to produce a sufficient number ofcells to treat a patient is reduced by at least 5% (and preferably atleast 10% or at least 20%) using DLD instead of Ficoll centrifugationand the CAR T cells can be prepared without the need for freezing. In apreferred method, cells are collected from a patient, processed by DLDand, optionally, an affinity method at the same site. Genetictransformation may also take place at the site and, preferably, no morethan one hour elapses from the time that apheresis is completed untilDLD is begun.

The invention also encompasses a method for decreasing the ratio ofplatelets to leukocytes in an apheresis sample by performingdeterministic lateral displacement (DLD) on the sample. DLD is carriedout in the absence of centrifugation or elutriation, to obtain a productin which the total number of platelets is at least 70% (and preferablyat least 90%) lower than in the apheresis sample. DLD should preferablybe carried out in a buffer that does not comprise intercalators and thatdoes not promote platelet aggregation. Preferably, the buffer does notcomprise dextran or other highly charge polymers.

The invention is not limited to leukocytes but also includes othertherapeutically valuable cells, especially cells that may be present inapheresis preparations, including circulating stem cells. The benefitsof DLD, including benefits due to the removal unwanted platelets, shouldapply to a wide variety of processes.

Methods of Using DLD for Large Volumes of Leukapheresis Material

One advantage of DLD is that it can be used to process small quantitiesof material with little increase in volume as well as relatively largequantities of material.

The procedure may be used on leukapheresis products that have a smallvolume due to the concentration of leukocytes by centrifugation as wellas in processing a large volume of material.

Thus, in another aspect, the invention is directed to a system forpurifying cells from large volume leukapheresis processes in which atleast one microfluidic device is used that separates materials by DLD.The objective is to obtain leukocytes that may be used therapeuticallyor that secrete agents that may be used therapeutically. Of particularimportance, the invention includes binding specific types of leukocytesto one or more carriers in a way that promotes and, optionally, alsocomplements DLD separation and then performing DLD on the complex. Inthis way, specific types of leukocytes may be separated from cells thatare about the same size and that, in the absence of complex formation,could not be resolved by DLD. In this regard, a two step procedure asdiscussed above may sometimes be advantageous in which a one DLDprocedure separates unbound leukocytes from smaller material and aanother DLD procedure separates a carrier-leukocyte complex fromuncomplexed cells. Essentially the same technique can be used in othercontexts as well, e.g., on cultured cells, provided that cell specificcarriers are available. In all instances, the cells may be recombinantlygenetically engineered to alter the expression of one or more of theirgenes.

For leukapheresis material, the microfluidic devices must have at leastone channel extending from a sample inlet to both a “collection outlet”for recovering white blood cells (WBCs) or specific leukocyte-carriercomplexes and a “waste outlet” through which material of a differentsize (generally smaller) than WBCs or uncomplexed leukocytes flow. Thechannel is bounded by a first wall and a second wall opposite from thefirst wall and includes an array of obstacles arranged in rows, witheach successive row being shifted laterally with respect to a previousrow. The obstacles are disposed in a manner such that, whenleukapheresis material is applied to an inlet of the device andfluidically passed through the channel, cells or cell complexes aredeflected to the collection outlet (or outlets) where an enrichedproduct is collected and material of a different (generally smaller)size flows to one or more separate waste outlets.

In order to facilitate the rapid processing of large volumes of startingmaterial, the obstacles in microfluidic devices may be designed in theshape of diamonds or triangles and each device may have 6-40 channels.In addition, the microfluidic devices may be part of a system comprising2-20 microfluidic devices (see FIG. 7). Individual devices may beoperated at flow rates of 14 ml/hr but flow rates of at least 25 ml/hr(preferably at least 40, 60, 80 or 100 ml per hour) are preferable andallow large sample volumes (at least 200 ml and preferably 400-600 ml)to be processed within an hour.

Separation of Viable Cells

In another aspect, the invention is directed to methods of separating aviable cell from a nonviable cell comprising: (a) obtaining a samplecomprising the viable cell and the nonviable cell, where the viable cellcan have a first predetermined size and the nonviable cell can have asecond predetermined size; and where the first predetermined size can begreater than or equal to a critical size, and the second predeterminedsize can be less than the critical size; (b) applying the sample to adevice, where the device can comprise an array of obstacles arranged inrows, where the rows can be shifted laterally with respect to oneanother, where the rows can be configured to deflect a particle greaterthan or equal to the critical size in a first direction and a particleless than the critical size in a second direction; and (c) flowing thesample through the device, where the viable cell can be deflected by theobstacles in the first direction, and the non-viable cell can bedeflected in the second direction, thereby separating the viable cellfrom the nonviable cell. The critical size can be about 1.1-fold greaterthan the second predetermined size and in some embodiments, the viablecell can be an actively dividing cell. In some embodiments, the devicecan comprise at least three zones with progressively smaller obstaclesand gaps.

Separation of Adherent Cells

The invention also includes a method of obtaining adherent target cells,preferably cells of therapeutic value, e.g., adherent stem cells, by: a)obtaining a crude fluid composition comprising the adherent target cellsfrom a patient; and b) performing Deterministic Lateral Displacement(DLD) to obtain a composition enriched in the adherent target cells.During this process, the adherent target cells may be bound to one ormore carriers in a way that promotes or complements DLD separation. Forexample carriers may have on their surface an affinity agent (e.g., anantibody, activator, hapten or aptamer) that binds with specificity (asdefined above) to the adherent target cells and may be transfected ortransduced with nucleic acids designed to impart on the cells a desiredphenotype, e.g., to express a chimeric molecule (preferably a proteinthat makes the cells of greater therapeutic value).

Carriers may be added at the time that the crude fluid composition isbeing collected or, alternatively after collection is completed butbefore DLD is performed for the first time. In a second alternative, DLDmay be performed for a first time before carrier is added. For example,if the adherent cell has a size less than the critical size, the crudefluid composition may be applied to the device before the carrier isadded, the adherent cell may be recovered, the cells may then beattached to one or more carriers to form a complex that is larger thanthe critical size of a device, a second DLD step may then be performedand the carrier adherent cell complexes may be collected.

Preferably, no more than three hours (and more preferably no more thantwo hours, or one hour) elapse from the time that the obtaining of thecrude fluid composition from the patient is completed until the adherentcell is bound to a carrier for the first time. In another preferredembodiment, no more than four hours (and preferably no more than threeor two hours) elapse from the time that the obtaining of the crude fluidcomposition from the patient is complete until the first time that theadherent cell or a carrier adherent cell complex is collected from thedevice for the first time.

The methodology described above may be used to separate adherent targetcells, e.g., adherent stem cells, from a plurality of other cells. Themethod involves: a) contacting a crude fluid composition comprising theadherent target cells and the plurality of other cells, wherein theadherent target cells are at least partially associated with one or morecarriers in a way that promotes DLD separation and form carrierassociated adherent target cell complexes, wherein the complexescomprise an increased size relative to the plurality of other cells, andwherein the size of the carrier associated adherent cell complexes ispreferably at least 50% greater than a critical size, and other,uncomplexed cells comprise a size less than the critical size; b)applying the crude fluid composition containing the carrier associatedadherent cell complexes to a device, wherein the device comprises anarray of obstacles arranged in rows, wherein the rows are shiftedlaterally with respect to one another, wherein the rows are configuredto deflect cells or complexes greater than or equal to the critical sizein a first direction and cells or complexes less than the critical sizein a second direction; c) flowing the crude fluid composition comprisingthe carrier associated adherent target cell complexes through thedevice, wherein the complexes are deflected by the obstacles in thefirst direction, and uncomplexed cells are deflected in the seconddirection, thereby separating the carrier associated adherent cellcomplexes from the other uncomplexed cells; d) collecting a fluidcomposition comprising the separated carrier associated adherent targetcell complexes.

The diameter of the complex formed between adherent target cells and oneor more carriers should preferably be at least 20% larger than theuncomplexed cells and preferably at least 50% larger, at least twice aslarge or at least ten times as large. This increase in size may beeither due to the binding of a single large carrier to the adherenttarget cells or due to the binding of several smaller carriers. Bindingmay involve using: a) only carriers with a diameter at least as large(or in other embodiments, at least twice as large or at least ten timesas large) as that of the adherent target cells; b) only carriers with adiameter no more than 50% (or in other embodiments, no more than 25% or15%) as large as that of the adherent target cells; or c) mixtures oflarge and small carriers with these size characteristics (e.g., theremay be one group of carriers with a diameter at least as large (or atleast twice or ten times as large) as the adherent target cells and asecond group of carriers with a diameter no more than 50% (or no morethan 25% or 15%) as large as that of the adherent target cells.Typically a carrier will have a diameter of 1-1000 μm (and often in therange of 5-600 or 5-400 μm).

The carriers may be made of any of the materials that are known in theart for the culturing of adherent cells including polypropylene,polystyrene, glass, gelatin, collagen, polysaccharides, plastic,acrylamide and alginate. They may be uncoated or coated with materialsthat promote adhesion and growth (e.g., serum, collagen, proteins orpolymers) and may have agents (e.g., antibodies, antibody fragments,substrates, activators or other materials) attached to their surfaces.In some embodiments, the diluent can be growth media, the steps can beperformed sequentially and, after step (d), buffer exchange can beperformed.

Examples of specific adherent cells that may be isolated in the methodsdescribed above include: an MRC-5 cell; a HeLa cell; a Vero cell; an NIH3T3 cell; an L929 cell; a Sf21 cell; a Sf9 cell; an A549 cell; an A9cell; an AtT-20 cell; a BALB/3T3 cell; a BHK-21 cell; a BHL-100 cell; aBT cell; a Caco-2 cell; a Chang cell; a Clone 9 cell; a Clone M-3 cell;a COS-1 cell; a COS-3 cell; a COS-7 cell; a CRFK cell; a CV-1 cell; aD-17 cell; a Daudi cell; a GH1 cell; a GH3 cell; an HaK cell; an HCT-15cell; an HL-60 cell; an HT-1080 cell; a HEK cell, HT-29 cell; an HUVECcell; an I-10 cell; an IM-9 cell; a JEG-2 cell; a Jensen cell; a Jurkatcell; a K-562 cell; a KB cell; a KG-1 cell; an L2 cell; an LLC-WRC 256cell; a McCoy cell; a MCF7 cell; a WI-38 cell; a WISH cell; an XC cell;a Y-1 cell; a CHO cell; a Raw 264.7 cell; a HEP G2 cell; a BAE-1 cell;an SH-SY5Y cell, and any derivative thereof.

Separation of Cells Bound to an Activator

The invention also includes methods of purifying cells capable ofactivation using the procedures described above. In a preferredembodiment, the invention is directed to a method of separating anactivated cell from a plurality of other cells by: a) contacting a crudefluid composition comprising a cell capable of activation and theplurality of other cells with one or more carriers, in a way thatpromotes DLD separation, wherein one or more of the carriers comprise acell activator, wherein one or more carriers are at least partiallyassociated with the cell capable of activation by the cell activatorupon or after contact to generate a carrier associated cell, wherein theassociation of the cell activator with the cell capable of activation atleast partially activates the cell capable of activation, wherein thecarrier associated cell complex comprises an increased size relative toother cells, and wherein a size of the carrier associated cell complexis greater than or equal to a critical size, and the cells in theplurality of other cells comprise a size less than the critical size; b)applying the crude fluid composition to a device, wherein the devicecomprises an array of obstacles arranged in rows; wherein the rows areshifted laterally with respect to one another, wherein the rows areconfigured to deflect a particle greater than or equal to the criticalsize in a first direction and a particle less than the critical size ina second direction; c) flowing the sample through the device, whereinthe carrier associated cell complex is deflected by the obstacles in thefirst direction, and the cells in the plurality of other cells aredeflected in the second direction, thereby separating the activated cellfrom the other cells of the plurality. The fluid composition comprisingthe separated carrier associated cell complex may then be collected.During this process the cells may optionally be transfected ortransduced with nucleic acids designed to impart on the cells a desiredphenotype, e.g., to express a chimeric molecule (preferably a proteinthat makes the cells of greater therapeutic value).

The cell capable of activation may be selected from the group consistingof: a T cell, a B cell, a macrophage, a dendritic cell, a granulocyte,an innate lymphoid cell, a megakaryocyte, a natural killer cell, athrombocyte, a synoviocyte, a beta cell, a liver cell, a pancreaticcell; a DE3 lysogenized cell, a yeast cell, a plant cell, and a stemcell.

The cell activator may be selected from the group consisting of: anantibody or antibody fragment, CD3, CD28, an antigen, a helper T cell, areceptor, a cytokine, a glycoprotein, and any combination thereof. Inother embodiments, the activator may be a small compound and may beselected from the group consisting of insulin, IPTG, lactose,allolactose, a lipid, a glycoside, a terpene, a steroid, an alkaloid,and any combination thereof.

In a preferred embodiment, the cell capable of activation is collectedfrom a patient as part of a crude fluid composition comprising the cellcapable of activation and a plurality of other cells, wherein no morethan four hours (and preferably no more than three hours, two hours orone hour) elapse from the time that the obtaining of the crude fluidcomposition from the patient is completed until the cell capable ofactivation is bound to the carrier. It is also preferable that no morethan four hours elapse from the time that the obtaining of the crudefluid composition from the patient is completed until step c) iscompleted. Alternatively, the method may be altered by binding activatorbefore collection of cells begins.

Preferably, the diameter of the complex formed between a cell capable ofactivation and one or more carriers should be at least 20% larger thanthe uncomplexed cells and more preferably at least 50% larger, at leasttwice as large or at least ten times as large. This increase in size maybe either due to the binding of a single large carrier to the cellcapable of activation or due to the binding of several smaller carriers.Binding may involve using: a) only carriers with a diameter at least aslarge (or in other embodiments, at least twice as large or at least tentimes as large) as that of the cell capable of activation; b) onlycarriers with a diameter no more than 50% (or in other embodiments, nomore than 25% or 15%) as large as that of the cell capable ofactivation; or c) mixtures of large and small carriers with these sizecharacteristics (e.g., there may be one group of carriers with adiameter at least as large (or at least twice or ten times as large) asthe cell capable of activation and a second group of carriers with adiameter no more than 50% (or no more than 25% or 15%) as large as thatof the cell capable of activation. Typically a carrier will have adiameter of 1-1000 μm (and often in the range of 5-600 or 5-400 μm).

Separating Compounds from Cells

In another embodiment, the invention includes methods of removing acompound from a cell comprising: (a) obtaining a fluid compositioncomprising the cell and the compound, where the cell has a predeterminedsize that is greater than a predetermined size of the compound, andwhere the predetermined size of the cell is greater than or equal to acritical size, and the predetermined size of the compound is less thanthe critical size; (b) applying the sample to a device, where the devicecomprises an array of obstacles arranged in rows, where the rows areshifted laterally with respect to one another, where the rows areconfigured to deflect a particle greater than or equal to the criticalsize in a first direction and a particle less than the critical size ina second direction; and (c) flowing the sample through the device,during which the cell is deflected by the obstacles in the firstdirection, and the compound can be deflected in the second direction,thereby removing the compound from the cell. In some embodiments, themethod can further comprise culturing the cell after step (c) orrecycling the cells to a culture from which the fluid composition ofstep a) was obtained.

The compound may be a toxic compound and may be selected from the groupconsisting of: an antibiotic, an antifungal, a toxic metabolite, sodiumazide, a metal ion, an endotoxin, a plasticizer, a pesticide, and anycombination thereof. In other embodiments, the compound can be a spentchemical component.

Continuous Purification of a Secreted Cellular Product

The invention also includes methods of continuously purifying a secretedproduct from a cell comprising: (a) obtaining a fluid compositioncomprising the cell (which may be a cell culture composition), where thecell is suspended in the fluid composition (or the cell is bound to oneor more carriers in a way that promotes DLD separation and that forms acarrier-cell complex) and where the cell secretes the secreted productinto the fluid composition, where the cell (or the carrier-cell complex)has a predetermined size that is greater than a predetermined size ofthe secreted product, and where the predetermined size of the cell (orthe carrier-cell complex) is greater than or equal to a critical size,and the predetermined size of the secreted product is less than thecritical size; (b) applying the fluid composition comprising the cell(or the carrier-cell complex) to a device for DLD, where the devicecomprises an array of obstacles arranged in rows; where the rows areshifted laterally with respect to one another, where the rows areconfigured to deflect a particle greater than or equal to the criticalsize in a first direction and a particle less than the critical size ina second direction; (c) flowing the fluid composition comprising thecell or the carrier-cell complex through the device, where the cell orcarrier-cell complex is deflected by the obstacles in the firstdirection, and the secreted product is deflected in the seconddirection, thereby separating the secreted product from the cell; (d)collecting the secreted product, thereby producing a fluid compositionof the secreted product that is purified; (e) collecting a recoveredfluid composition comprising the separated cells or carrier-cellcomplexes; (f) re-applying the cells (or the carrier-cell complexes) tothe fluid composition; and repeating steps (a) through (e); therebycontinuously purifying the secreted product from the cell.

The secreted product can be a protein, an antibody, a biofuel, apolymer, a small molecule, and any combination thereof and the cell canbe a bacterial cell, an algae cell, a mammalian cell, and a tumor cell.In one preferred embodiment, the secreted product is a therapeuticallyvaluable protein, antibody, polymer or small molecule. In addition thefluid composition of step a) may be obtained from a culture in whichcells are grown on carriers.

Use of Microfluidic Sizing Devices

More broadly, the invention is directed to methods of engineering apopulation of target cells prepared by any size based microfluidicseparation method. Differences in sorting cells based on size may be theresult of using bump arrays as discussed herein or result from inertialforces generated by controlling the flow rate during separations orthrough the design of the microfluidic devices themselves (see U.S. Pat.Nos. 9,895,694 and 9,610,582, incorporated herein by reference in theirentirety). There may be only a single separation procedure used or theremay be more than one. For example, target cells may be separated fromsmaller particles and cells using one microfluidic procedure and fromlarger particles and cells using a second procedure.

Once target cells are isolated, they are genetically engineered to havea desired phenotype. This may be accomplished using standard recombinantmethodology for transfecting or transforming cells. For example, cellsmay be transfected with a vector to express a recombinant phenotype. Byavoiding centrifugation prior to genetic engineering, there should be atleast a 20% increase in cells with the desired characteristics.

Preferred target cells are leukocytes (especially T cells) or stem cellsand the preferred crude fluid composition is blood or an apheresispreparation obtained from a patient. A central objective is to reducethe ratio of platelets to target cells in these preparations by at least50% and preferably by at least 80% or 90%. The isolation of target cellsshould take place under conditions such that a product is obtained inwhich the total number of platelets is at least 70% (and preferably atleast 90%) lower than in the starting apheresis preparation.

During or after genetic engineering, cells are expanded in cell culture.Using the procedures described above, the number of T cells obtainedafter 14 days in culture should be at least two times (and preferably atleast five or ten times) higher than in a procedure in which cells areisolated using centrifugation. In addition, the percentage of memory Tcells in culture relative to the total number of T cells should be atleast 10% (and preferably 20% or 30%) higher than in a procedure inwhich T cells are isolated by centrifugation.

No more than one hour should elapse from the time that apheresiscollection is completed until the time that DLD is performed and no morethan four hours should elapse from the time the obtaining of theapheresis sample is completed until the target cells have been isolatedand are genetically engineered.

In a particularly preferred embodiment, the method described above isused for the production of CAR T cells. This involves first obtaining acrude fluid composition containing T cells by apheresis and thenisolating the cells on a microfluidic device using one or moreprocedures that separate T cells from platelets based on differences insize. As a result, a product should be obtained that is an enriched in Tcells and depleted in platelets. In the next step, the isolated T cellsare genetically engineered to express chimeric antigen receptors (CARs)on their surface. These cells are cultured to expand their number andthen collected. The T cells should not be centrifuged or elutriated atany step prior to being genetically engineered and, in a preferredembodiment, reagents used for genetic engineering are separated fromcells by size using a microfluidic device. In an additional preferredembodiment, T cells are collected by being transferred into apharmaceutical composition for administration to a patient.

T cells should not be frozen before being collected or transferred intoa pharmaceutical composition and preferably at least 90% of plateletsare removed. Prior to, or during, culturing, cells may be exposed to a Tcell activator or a carrier. This may help to stabilize the cells andmay also facilitate size-based microfluidic separation. It should benoted however, that neither activators nor the carriers necessarily needto be bound to magnetic beads or particles.

Compared to a procedure in which cells are isolated or concentrated bycentrifugation, CAR T cells obtained by microfluidic separation shouldbe available for use by a patient at least one day (and preferably, atleast 3, 5 or 10 days) earlier. Overall, the time necessary to produce asufficient number of CAR T cells for treatment should be at least 10%(and preferably at least 20% or 30%) shorter than when the same methodis carried out using Ficoll centrifugation to isolate cells. This ispartly because, by using a size based microfluidic separation, thenumber of CAR T cells obtained after 14 days in culture will typicallybe at least two times (and preferably four or eight times) higher thanthe number in cultures which use cells obtained by Ficollcentrifugation. In addition, when cells prepared by the present methodare administered to a patient, they should exhibit at least 10% lesssenescence than cells isolated from an apheresis composition bycentrifugation.

The present invention also encompasses treating a patient for a diseaseor condition by administering a therapeutically effective amount ofcells prepared by the methods discussed above. This includes any diseaseor condition that responds to engineered leukocytes or stem cells and,at least in the case of CAR T cells, cancer is among the diseases thatmay be treated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G: FIGS. 1A-C illustrate different operating modes of DLD.This includes: i) Separation (FIG. 1A), ii) Buffer Exchange (FIG. 1B)and iii) Concentration (FIG. 1C). In each mode, essentially allparticles above a critical diameter are deflected in the direction ofthe array from the point of entry, resulting in size selection, bufferexchange or concentration as a function of the geometry of the device.In all cases, particles below the critical diameter pass directlythrough the device under laminar flow conditions and subsequently offthe device. FIG. 1D shows a 14 lane DLD design used in separation mode.The full length of the depicted array and microchannel is 75 mm and thewidth is 40 mm, each individual lane is 1.8 mm across. FIGS. 1E-1F areenlarged views of the plastic diamond post array and consolidatingcollection ports for the exits. FIG. 1G depicts a photo of aleukapheresis product being processed using a prototype device at 10PSI.

FIGS. 2A-2H: FIG. 2A is a scatter plot showing the range of normal donorplatelet and WBC cell counts used in this study. Mean counts of WBC:162.4×10⁶/mL and Platelets: 2718×10³/μL respectively (+). The outliersample (▴), clogged the 20 μm prefilter and was excluded from the dataset. Input sample shown (FIGS. 2C and 2D). Representative 24-hour oldnormal donor leukapheresis input (FIG. 2B) and PBMC product processed byeither a 14-lane diamond post DLD at 10 PSI (FIG. 2E) or Ficoll-Hypaque(FIG. 2F). Representative DLD product (FIG. 2G) and Ficoll (FIG. 2H)from the same Leukapheresis donor (#37). Input (FIGS. 2B, 2C, 2D) andproduct fractions (FIGS. 2E and 2F) were fixed and stained on slideswith CD41-FITC (platelets) plus CD45-Alexa647 (WBC) and counter-stainedwith DAPI (nuclear DNA).

FIG. 3: This figure concerns the consistency of cell activation in DLDvs. Ficoll and Direct Magnet approaches (CD4, CD8 vs CD25 Day 8). Cellactivation and Phenotypic profile shows a shift during expansion towardsclassic central memory T cell associated phenotype (Day 8). Cells werecounted and de-beaded as described previously. At each time point˜100,000 cells were stained with CD3-BV421, CD45RA-BV605, CD95-FITC,CD279-PE, CD25-APC, CD4-Alexa 700, and CD8-APC-Cy7, incubated for 30 atroom temperature in the dark and washed with 10 volumes of PBS prior tocentrifugation and fixation in 1.0% Para-formaldehyde in PBS. Sampleswere acquired on a BD FACSAria, and analyzed using a CD3 and forward andside scatter gate using FlowLogic software.

FIG. 4 is a graph depicting rapid gain of memory cell phenotype andconsistent activation of samples via DLD compared to Ficoll & DirectMagnet. Plot of % CD45RA−, CD25+ cells measuring conversion to T cellactivation and conversion via CD45 RO is status shown. Cells were fed200 Units IL-2/mL culture at Day 3 and again at day 8 only as theexperiment was designed to address initial ability to expand.

FIGS. 5A-5C: These figures concern the Fold Expansion of CD3 cells(×10⁶) from DLD, Ficoll and Direct Magnet. Aliquots of DLD product andFicoll cells were incubated with CD3/CD28 beads following Thermo-FisherCTS protocol using a T cell density of 1×10⁷ T cells/mL. A ratio of ˜2.5Beads/T cell and for the Direct Magnet using ˜5.0 Beads/T cell was used,cells and beads were incubated on a rotary mixer for 60 min prior tomagnetic separation. Either stimulated or unstimulated (unseparatedPMBC) cells were diluted in complete media (RPMI-1640+10%FBS+antibiotics without IL-2) to 0.5×10⁶/mL and were plated in timepoint specific reactions to avoid any disturbance of the cultures atintermediate time points. On Day 3, 200 IU of IL-2/mL was added to thestimulated and separated arm per manufacturer's recommendation. Cellcounts were determined on Day 3, 8, 15 after de-beading usingmanufacturers protocol (pipetting) by Coulter count (Scepter) andverified by bead based absolute counting using flow cytometry on a BDFACSCalibur using a no-wash approach with a fluorescence threshold onCD45 and staining with CD3− FITC, CD45−PerCP and using the DNA stainDRAQ5 to ensure effective discrimination of doublets and any cells withbeads still attached. Correlation between counting methods wasacceptable with a slope of 0.95, R2=0.944. Media was added to thecultures to maintain cell densities in an acceptable range (<3.0×10⁶/mL)on Days 6, and 9. Day 15 data point for the donor 21 was lost due tocontamination. Averages or % CV's shown in horizontal bars as indicated(FIG. 5A). FIG. 5B shows the percentage of T central memory cells (day15) and FIG. 5C shows the number of T central memory cells (day 15).

FIGS. 6A-6B concern cytometric analysis of T central memory cells andthe number of central memory cells produced. FIG. 6A: T Central MemoryCells: CD3+ T cells were gated on a singlet gate followed by a CD3 vSide scatter and central memory phenotyping using 4 parameter gate ofCD45RO, CCR7, CD28 and CD95 to define the central memory population. Thepopulation was back gated to display central memory cells, which in acolor figure are red, as fraction of T cells. Non-red cells in a colorfigure represent all non central memory T cells. FIG. 6B: PhenotypeConversion and Key Metrics (Day 15): Key metrics show # of donors wherethe number of central memory cells is >50%, with the average and % CVassociated with the central memory expansion.

FIG. 7 is a schematic showing how current individual chips have beendesigned to be stackable in layers to achieve throughput as demanded byany particular application using established manufacturing approaches.Injection molded layers are planned as systems are developed.

FIGS. 8A-8C: These are supplemental figures showing the concentration ofWBC via DLD. FIG. 8A: DLD Product Derived from Whole Blood: Whole bloodwas passed over first DLD to remove erythrocytes. A second, in line,concentrating DLD, designed to achieve a concentration factor of 12, wasconnected to the product output of the separating DLD. Equal volumes ofproduct and waste were added to tubes with equal numbers of absolutecount beads and analyzed by flow cytometry. The resulting relativecell:bead ratio for Waste (FIG. 8B) and for Concentrate (FIG. 8C) wascalculated compared to the input material to determine foldconcentration. Leukocytes were stained with CD45 PerCP and 1 mM DRAQ5,which was used as a fluorescence threshold to acquire both the beads andthe leukocytes. 5000 bead events acquired. (all reagents eBioscience).Designed Concentration Factor: 12.0×; Observed relative concentration:15.714/1.302=12.07×

FIG. 9 is a supplemental figure on the expression of CD25 and CD4 onunstimulated CD3+ T Cells purified by either DLD or Ficoll methods (Day8). Cells were prepared as described and analyzed as in FIG. 3. MeanCD4+25+: Ficoll: 20.25%: DLD: 8%.

FIG. 10: This is a supplemental figure on the allocation of IL-2expanded central memory T cells by major subsets. In the originalfigures: CD8 (Green), CD4 (Blue), CD4+CD8+(Red) Central memory cellswere sequentially gated: CD3+, CD45RO+CCR7+, CD28+CD95+. Relativeabundance of CD4 subset driven by IL2 is evident.

FIG. 11 is a supplemental figure depicting estimates of the number ofcentral memory T cells, post expansion with IL-2, assuming yields inthis study and a typical leukapheresis harvest from a donor with 50×10⁶WBC cells per/mL and containing 50% CD3 lymphocytes in 250 mL.

FIG. 12 illustrates a protocol that might, in principle, be used forproducing CAR T cells and administering the cells to a patient. It hasbeen included to contrast other procedures discussed herein and does notrepresent work actually performed.

FIG. 13 illustrates a proposed protocol for producing CAR T cells thatdiffers from the protocol of FIG. 12 in the initial steps of theprocedure. The steps in the center portion of the figure are includedfor purposes of comparison. The diagram is intended to illustrateinventive concepts and does not represent work actually performed.

FIG. 14 illustrates a second proposed protocol for producing CAR T cellsthat differs from the protocol of FIG. 12 in the initial steps of theprocedure. The steps in the center portion of the figure are includedfor purposes of comparison. As with FIGS. 12 and 13, the diagram isintended to illustrate inventive concepts and does not represent workactually performed.

FIG. 15 shows a schematic of a device for removing secreted productsfrom spent cells.

FIG. 16 shows a schematic of a device for continuous removal of toxiccompounds from actively growing cells.

FIG. 17 shows a schematic of a device for continuous removal of toxiccompounds from actively growing cells with the option of adding carriersbetween each iteration.

FIGS. 18A and 18B: FIG. 18 A shows an example of a mirrored array ofobstacles with a downshift. A central channel is between an array ofobstacles on the left and on the right. The central channel can be acollection channel for particles of at least a critical size (i.e.,particles of at least a critical size can be deflected by the arrays tothe central channels, whereas particles of less than the critical sizecan pass through the channel with the bulk flow). By downshifting rows,changes in the width of the channel relative to a mirrored array with adownshift can be achieved. The amount of downshift can vary based on thesize and/or cross-sectional shape of the obstacles. FIG. 18B illustratesa mirrored array of obstacles with no downshift. An array on the leftand an array on the right can deflect particles of at least a criticalsize to the central channel.

FIGS. 19A and 19B: These figures show the platelets remaining in anapheresis sample processed using DLD (FIG. 19A) and the plateletsremaining in an apheresis sample processed using Ficoll centrifugation(FIG. 19B). The platelets are the smaller, brighter cells (some of whichare shown by arrows) and leukocytes are the larger darker cells. It canbe seen that the relative number of platelets is substantially lower incells processed by DLD.

FIG. 20 graphically shows the percentage of platelets left in apheresissamples processed by Ficoll centrifugation (white bars) and apheresissamples processed by DLD (striped bars) using three different buffers.The X axis is the percentage of platelets remaining. Results for a 1%BSA/PBS buffer are shown on the far left side of the figure; percentagesfor a buffer containing an F127 poloxamer intercalator are in the centerand percentages for an elutriation buffer are on the far right.

FIG. 21 shows the results of FIG. 22 except that the Y axis representsthe number of platelets per leukocyte, i.e., the ratio of platelets toleukocytes. Bars with triplet stripes represent the ratio present in theinitial apheresis sample, white bars are the ratio after processing byFicoll centrifugation and bars with widely spaced single stripes are theratio after processing by DLD.

FIG. 22 is a bar graph showing the expansion of T cells that occurs asthe result of processing apheresis samples by Ficoll centrifugation(solid white bars) and DLD (bars with single stripes adjacent to whitebars). Moving from left to right the bars show the effect of adding 10%(bars with double stripes), 50% (stippled bars) and 100% (bars with darkwidely spaced bars adjacent to stippled bars) of the plateletsoriginally present in the apheresis starting material back to the cellsprocessed by DLD before expansion. The left side of the figure depictscells processed via DLD without EDTA and then stimulated with CD3/CD28at day 0, with CD3 positive cells counted on day 3, 7 and 14. The rightside of the figure shows cells processed with 2 mM EDTA and thenstimulated with CD3/CD28 at day 0, with CD3 positive cells counted onday 3, 7 and 14.

FIG. 23 is a comparison of variability in the expansion of T cellsobtained using three methods of processing: separation using magneticbeads alone (bar on the far left of the figure), separation using Ficollcentrifugation followed by magnetic beads (center bar), and separationby DLD followed by magnetic beads (bar on the far right).

FIG. 24 shows the percentage of cells present in the expanded T cellpopulations of FIG. 23 that are central memory T cells.

Overview of Workflow Figures: FIGS. 25-32 are all included to illustrateconcepts and do not concern any experiments actually performed. Thefigures illustrate the basic workflow typically involved in obtainingcells from a patient which are then processed and used therapeutically,typically to treat the same patient from which the cells were obtained.The figures, for the most part, refer specifically to the making of CART cells but it will be understood that the processes can be appliedgenerally to the making of leukocytes and other cells for therapeuticpurposes. In the case of many such processes, including those for CAR Tcells, the cells collected from patients are recombinantly engineered toexpress genes of therapeutic interest. Any type of such engineering maybe a part of the illustrated processes. The figures divide steps intothose that are performed at a clinic and those that are performed at aseparate site, which for the purposes of illustration has been termed a“manufacturing site.” FIG. 25 shows the workflow that might be expectedfor a typical process used, for example, in making CAR T cells.Transformation of the cells would typically occur at the manufacturingsite after cryopreserved samples are thawed, washed, debulked andenriched. FIG. 26 shows several places (stippled boxes) in a prototypeCAR T cell process where DLD might be used while maintaining the samebasic workflow. FIGS. 27-32 then present a number of illustrativeexamples of how DLD may be used to modify workflow and improve theprotoype process. Primary objectives are to produce cells of betteroverall quality, shorten the time needed to obtain a sufficient numberof cells to treat a patient and to automate steps that are currentlymore labor intensive.

FIG. 25 illustrates the basic workflow generally involved in currentmethods for processing cells from a patient for therapeutic use. Asillustrated, the process starts at a clinic with the collection of cells(“apheresis collection”), proceeds to a manufacturing site where thecells are expanded and may be engineered, and ends back at the clinicwith the administration of the cells to a patient (“Re-infusion”).

FIG. 26 shows several steps in the CAR T workflow where DLD could beused (stippled boxes). In this example, the basic workflow remainsessentially unchanged.

FIG. 27 illustrates that DLD may be used immediately after apheresis toclean up cells prior to freezing (stippled box). The advantages of usingDLD at this point are that it removes platelets from cells beforeshipment and thereby eliminates deleterious effects from plateletactivation, clot formation, any storage related degranulation andoverall loss of cells. In this manner, DLD improves the quality of cellcompositions being shipped and replaces one of the hands on labor stepsin the process with an automated counterpart.

FIG. 28: In this scenario, DLD is used during early steps in which cellsare cleaned up, activated, DLD separated and transfer into a desiredmedium, e.g., a growth medium (stippled boxes). Importantly, freezingand cryopreservation at the clinic and subsequent thawing at themanufacturing site are avoided which should reduce the time needed tocomplete the process, e.g., by about 2-3 days. Although not preferred, acryopreservation step could, if desired, still be performed. Other stepsthat might be avoided are indicated by crossed through text.

FIG. 29: In this scenario, DLD is used in conjunction with magneticbeads that bind to cell surface CD3 on T cells. This scenario has thesame advantage of reducing platelets described above but may be used toactivate cells and begin growth much sooner than in the prototypeprocedure. Cells may also be shipped at warm temperatures compatiblewith growth. As a result, counterpart steps that would have beenperformed at the manufacturing site are eliminated and overallprocessing time is correspondingly reduced.

FIG. 30: The most important difference in the scenario shown in FIG. 30compared to the scenario in FIG. 29 is that cells are geneticallytransformed with virus immediately after exposure to activator (seestippled boxes). Rather than several days elapsing before activation andtransformation, cells are purified, activated, transformed and growingin culture medium within 24 hours after apheresis collection iscompleted. Preferably the time from the completion of apheresis untilexposure to vector should be no more than 12 hours, and more preferably,no more than six or three hours. Most preferably, exposure would occurwithin two hours after apheresis is complete and, in all cases, beforecells are frozen. The figure shows many processing steps at themanufacturing site that may be eliminated and it is expected that thetime needed to obtain sufficient cells for treating a patient would bereduced by at least 3 or 4 days.

FIG. 31: The scenario shown in FIG. 31 is similar to that in FIG. 30except that separation by an affinity procedure (exemplified by magneticbeads that recognize CD3 containing cells) is added in the early stepsto help in the isolation of T cells. DLD is also used to purify and/orconcentrate cells.

FIG. 32: In the scenario shown in FIG. 32, T cells collected byapheresis are purified, engineered, expanded and concentrated forreinfusion without ever undergoing a step in which they are frozen. Allsteps can, if desired, be performed at the site where cells arecollected without a need for shipping.

DEFINITIONS

Apheresis: As used herein this term refers to a procedure in which bloodfrom a patient or donor is separated into its components, e.g., plasma,white blood cells and red blood cells. More specific terms are“plateletpheresis” (referring to the separation of platelets) and“leukapheresis” (referring to the separation of leukocytes). In thiscontext, the term “separation” refers to the obtaining of a product thatis enriched in a particular component compared to whole blood and doesnot mean that absolute purity has been attained.

CAR T cells: The term “CAR” is an acronym for “chimeric antigenreceptor.” A “CAR T cell” is therefore a T cell that has beengenetically engineered to express a chimeric receptor.

CAR T cell therapy: This term refers to any procedure in which a diseaseis treated with CAR T cells. Diseases that may be treated includehematological and solid tumor cancers, autoimmune diseases andinfectious diseases.

Carrier: As used herein, the term “carrier” refers an agent, e.g., abead, or particle, made of either biological or synthetic material thatis added to a preparation for the purpose of binding directly orindirectly (i.e., through one or more intermediate cells, particles orcompounds) to some or all of the compounds or cells present. Carriersmay be made from a variety of different materials, includingDEAE-dextran, glass, polystyrene plastic, acrylamide, collagen, andalginate and will typically have a size of 1-1000 μm. They may be coatedor uncoated and have surfaces that are modified to include affinityagents (e.g., antibodies, activators, haptens, aptamers, particles orother compounds) that recognize antigens or other molecules on thesurface of cells. The carriers may also be magnetized and this mayprovide an additional means of purification to complement DLD and theymay comprise particles (e.g., Janus or Strawberry-like particles) thatconfer upon cells or cell complexes non-size related secondaryproperties. For example the particles may result in chemical,electrochemical, or magnetic properties that can be used in downstreamprocesses, such as magnetic separation, electroporation, gene transfer,and/or specific analytical chemistry processes. Particles may also causemetabolic changes in cells, activate cells or promote cell division.

Carriers that bind “in a way that promotes DLD separation”: This term,refers to carriers and methods of binding carriers that affect the waythat, depending on context, a cell, protein or particle behaves duringDLD. Specifically, “binding in a way that promotes DLD separation” meansthat: a) the binding must exhibit specificity for a particular targetcell type, protein or particle; and b) must result in a complex thatprovides for an increase in size of the complex relative to the unboundcell, protein or particle. In the case of binding to a target cell,there must be an increase of at least 2 μm (and alternatively at least20, 50, 100, 200, 500 or 1000% when expressed as a percentage). In caseswhere therapeutic or other uses require that target cells, proteins orother particles be released from complexes to fulfill their intendeduse, then the term “in a way that promotes DLD separation” also requiresthat the complexes permit such release, for example by chemical orenzymatic cleavage, chemical dissolution, digestion, due to competitionwith other binders, or by physical shearing (e.g., using a pipette tocreate shear stress) and the freed target cells, proteins or otherparticles must maintain activity; e.g., therapeutic cells after releasefrom a complex must still maintain the biological activities that makethem therapeutically useful.

Carriers may also bind “in a way that complements DLD separation”: Thisterm refers to carriers and methods of binding carriers that change thechemical, electrochemical, or magnetic properties of cells or cellcomplexes or that change one or more biological activities of cells,regardless of whether they increase size sufficiently to promote DLDseparation. Carriers that complement DLD separation also do notnecessarily bind with specificity to target cells, i.e., they may haveto be combined with some other agent that makes them specific or theymay simply be added to a cell preparation and be allowed to bindnon-specifically. The terms “in a way that complements DLD separation”and “in a way that promotes DLD separation” are not exclusive of oneanother. Binding may both complement DLD separation and also promote DLDseparation. For example a polysaccharide carrier may have an activatoron its surface that increases the rate of cell growth and the binding ofone or more of these carriers may also promote DLD separation.Alternatively binding may just promote DLD separation or just complementDLD separation.

Target cells: As used herein “target cells” are the cells that variousprocedures described herein require or are designed to purify, collect,engineer etc. What the specific cells are will depend on the context inwhich the term is used. For example, if the objective of a procedure isto isolate a particular kind of stem cell, that cell would be the targetcell of the procedure.

Isolate, purify: Unless otherwise indicated, these terms, as usedherein, are synonymous and refer to the enrichment of a desired productrelative to unwanted material. The terms do not necessarily mean thatthe product is completely isolated or completely pure. For example, if astarting sample had a target cell that constituted 2% of the cells in asample, and a procedure was performed that resulted in a composition inwhich the target cell was 60% of the cells present, the procedure wouldhave succeeded in isolating or purifying the target cell.

Bump Array: The terms “bump array” and “obstacle array” are usedsynonymously herein and describe an ordered array of obstacles that aredisposed in a flow channel through which a cell or particle-bearingfluid can be passed.

Deterministic Lateral Displacement: As used herein, the term“Deterministic Lateral Displacement” or “DLD” refers to a process inwhich particles are deflected on a path through an array,deterministically, based on their size in relation to some of the arrayparameters. This process can be used to separate cells, which isgenerally the context in which it is discussed herein. However, it isimportant to recognize that DLD can also be used to concentrate cellsand for buffer exchange. Processes are generally described herein interms of continuous flow (DC conditions; i.e., bulk fluid flow in only asingle direction). However, DLD can also work under oscillatory flow (ACconditions; i.e., bulk fluid flow alternating between two directions).

Critical size: The “critical size” or “predetermined size” of particlespassing through an obstacle array describes the size limit of particlesthat are able to follow the laminar flow of fluid. Particles larger thanthe critical size can be ‘bumped’ from the flow path of the fluid whileparticles having sizes lower than the critical size (or predeterminedsize) will not necessarily be so displaced. When a profile of fluid flowthrough a gap is symmetrical about the plane that bisects the gap in thedirection of bulk fluid flow, the critical size can be identical forboth sides of the gap; however when the profile is asymmetrical, thecritical sizes of the two sides of the gap can differ.

Fluid flow: The terms “fluid flow” and “bulk fluid flow” as used hereinin connection with DLD refer to the macroscopic movement of fluid in ageneral direction across an obstacle array. These terms do not take intoaccount the temporary displacements of fluid streams for fluid to movearound an obstacle in order for the fluid to continue to move in thegeneral direction.

Tilt angle ε: In a bump array device, the tilt angle is the anglebetween the direction of bulk fluid flow and the direction defined byalignment of rows of sequential (in the direction of bulk fluid flow)obstacles in the array.

Array Direction: In a bump array device, the “array direction” is adirection defined by the alignment of rows of sequential obstacles inthe array. A particle is “bumped” in a bump array if, upon passingthrough a gap and encountering a downstream obstacle, the particle'soverall trajectory follows the array direction of the bump array (i.e.,travels at the tilt angle ε relative to bulk fluid flow). A particle isnot bumped if its overall trajectory follows the direction of bulk fluidflow under those circumstances.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is primarily concerned with the use of DLD inpreparing cells that are of therapeutic value. The text below providesguidance regarding methods disclosed herein and information that may aidin the making and use of devices involved in carrying out those methods.

I. Designing Microfluidic Plates

Cells, particularly cells in compositions prepared by apheresis orleukapheresis, may be isolated by performing DLD using microfluidicdevices that contain a channel through which fluid flows from an inletat one end of the device to outlets at the opposite end. Basicprinciples of size based microfluidic separations and the design ofobstacle arrays for separating cells have been provided elsewhere (see,US 2014/0342375; US 2016/0139012; U.S. Pat. Nos. 7,318,902 and7,150,812, which are hereby incorporated herein in their entirety) andare also summarized in the sections below.

During DLD, a fluid sample containing cells is introduced into a deviceat an inlet and is carried along with fluid flowing through the deviceto outlets. As cells in the sample traverse the device, they encounterposts or other obstacles that have been positioned in rows and that formgaps or pores through which the cells must pass. Each successive row ofobstacles is displaced relative to the preceding row so as to form anarray direction that differs from the direction of fluid flow in theflow channel. The “tilt angle” defined by these two directions, togetherwith the width of gaps between obstacles, the shape of obstacles, andthe orientation of obstacles forming gaps are primary factors indetermining a “critical size” for an array. Cells having a size greaterthan the critical size travel in the array direction, rather than in thedirection of bulk fluid flow and particles having a size less than thecritical size travel in the direction of bulk fluid flow. In devicesused for leukapheresis-derived compositions, array characteristics maybe chosen that result in white blood cells being diverted in the arraydirection whereas red blood cells and platelets continue in thedirection of bulk fluid flow. In order to separate a chosen type ofleukocyte from others having a similar size, a carrier may then be usedthat binds to that cell with in a way that promotes DLD separation andwhich thereby results in a complex that is larger than uncomplexedleukocytes. It may then be possible to carry out a separation on adevice having a critical size smaller than the complexes but bigger thanthe uncomplexed cells.

The obstacles used in devices may take the shape of columns or betriangular, square, rectangular, diamond shaped, trapezoidal, hexagonalor teardrop shaped. In addition, adjacent obstacles may have a geometrysuch that the portions of the obstacles defining the gap are eithersymmetrical or asymmetrical about the axis of the gap that extends inthe direction of bulk fluid flow.

II. Making and Operating Microfluidic Devices

General procedures for making and using microfluidic devices that arecapable of separating cells on the basis of size are well known in theart. Such devices include those described in U.S. Pat. Nos. 5,837,115;7,150,812; 6,685,841; 7,318,902; 7,472,794; and 7,735,652; all of whichare hereby incorporated by reference in their entirety. Other referencesthat provide guidance that may be helpful in the making and use ofdevices for the present invention include: U.S. Pat. Nos. 5,427,663;7,276,170; 6,913,697; 7,988,840; 8,021,614; 8,282,799; 8,304,230;8,579,117; US 2006/0134599; US 2007/0160503; US 20050282293; US2006/0121624; US 2005/0266433; US 2007/0026381; US 2007/0026414; US2007/0026417; US 2007/0026415; US 2007/0026413; US 2007/0099207; US2007/0196820; US 2007/0059680; US 2007/0059718; US 2007/005916; US2007/0059774; US 2007/0059781; US 2007/0059719; US 2006/0223178; US2008/0124721; US 2008/0090239; US 2008/0113358; and WO2012094642 all ofwhich are also incorporated by reference herein in their entirety. Ofthe various references describing the making and use of devices, U.S.Pat. No. 7,150,812 provides particularly good guidance and U.S. Pat. No.7,735,652 is of particular interest with respect to microfluidic devicesfor separations performed on samples with cells found in blood (in thisregard, see also US 2007/0160503).

A device can be made using any of the materials from which micro- andnano-scale fluid handling devices are typically fabricated, includingsilicon, glasses, plastics, and hybrid materials. A diverse range ofthermoplastic materials suitable for microfluidic fabrication isavailable, offering a wide selection of mechanical and chemicalproperties that can be leveraged and further tailored for specificapplications.

Techniques for making devices include Replica molding, Softlithographywith PDMS, Thermoset polyester, Embossing, Injection Molding, LaserAblation and combinations thereof. Further details can be found in“Disposable microfluidic devices: fabrication, function and application”by Fiorini, et al. (BioTechniques 38:429-446 (March 2005)), which ishereby incorporated by reference herein in its entirety. The book “Labon a Chip Technology” edited by Keith E. Herold and Avraham Rasooly,Caister Academic Press Norfolk UK (2009) is another resource for methodsof fabrication, and is hereby incorporated by reference herein in itsentirety.

High-throughput embossing methods such as reel-to-reel processing ofthermoplastics is an attractive method for industrial microfluidic chipproduction. The use of single chip hot embossing can be a cost-effectivetechnique for realizing high-quality microfluidic devices during theprototyping stage. Methods for the replication of microscale features intwo thermoplastics, polymethylmethacrylate (PMMA) and/or polycarbonate(PC), are described in “Microfluidic device fabrication by thermoplastichot-embossing” by Yang, et al. (Methods Mol. Biol. 949: 115-23 (2013)),which is hereby incorporated by reference herein in its entirety

The flow channel can be constructed using two or more pieces which, whenassembled, form a closed cavity (preferably one having orifices foradding or withdrawing fluids) having the obstacles disposed within it.The obstacles can be fabricated on one or more pieces that are assembledto form the flow channel, or they can be fabricated in the form of aninsert that is sandwiched between two or more pieces that define theboundaries of the flow channel.

The obstacles may be solid bodies that extend across the flow channel,in some cases from one face of the flow channel to an opposite face ofthe flow channel. Where an obstacle is integral with (or an extensionof) one of the faces of the flow channel at one end of the obstacle, theother end of the obstacle can be sealed to or pressed against theopposite face of the flow channel. A small space (preferably too smallto accommodate any particles of interest for an intended use) istolerable between one end of an obstacle and a face of the flow channel,provided the space does not adversely affect the structural stability ofthe obstacle or the relevant flow properties of the device.

The number of obstacles present should be sufficient to realize theparticle-separating properties of the arrays. The obstacles cangenerally be organized into rows and columns (Note: Use of the term“rows and columns” does not mean or imply that the rows and columns areperpendicular to one another). Obstacles that are generally aligned in adirection transverse to fluid flow in the flow channel can be referredto as obstacles in a column. Obstacles adjacent to one another in acolumn may define a gap through which fluid flows.

Obstacles in adjacent columns can be offset from one another by a degreecharacterized by a tilt angle, designated ε (epsilon). Thus, for severalcolumns adjacent to one another (i.e., several columns of obstacles thatare passed consecutively by fluid flow in a single direction generallytransverse to the columns), corresponding obstacles in the columns canbe offset from one another such that the corresponding obstacles form arow of obstacles that extends at the angle ε relative to the directionof fluid flow past the columns. The tilt angle can be selected and thecolumns can be spaced apart from each other such that 1/ε (whenexpressed in radians) is an integer, and the columns of obstacles repeatperiodically. The obstacles in a single column can also be offset fromone another by the same or a different tilt angle. By way of example,the rows and columns can be arranged at an angle of 90 degrees withrespect to one another, with both the rows and the columns tilted,relative to the direction of bulk fluid flow through the flow channel,at the same angle of ε.

Surfaces can be coated to modify their properties and polymericmaterials employed to fabricate devices, can be modified in many ways.In some cases, functional groups such as amines or carboxylic acids thatare either in the native polymer or added by means of wet chemistry orplasma treatment are used to crosslink proteins or other molecules. DNAcan be attached to COC and PMMA substrates using surface amine groups.Surfactants such as Pluronic® can be used to make surfaces hydrophilicand protein repellant by adding Pluronic® to PDMS formulations. In somecases, a layer of PMMA is spin coated on a device, e.g., microfluidicchip and PMMA is “doped” with hydroxypropyl cellulose to vary itscontact angle.

To reduce non-specific adsorption of cells or compounds, e.g., releasedby lysed cells or found in biological samples, onto the channel walls,one or more walls may be chemically modified to be non-adherent orrepulsive. The walls may be coated with a thin film coating (e.g., amonolayer) of commercial non-stick reagents, such as those used to formhydrogels. Additional examples of chemical species that may be used tomodify the channel walls include oligoethylene glycols, fluorinatedpolymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid,bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA,methacrylated PEG, and agarose. Charged polymers may also be employed torepel oppositely charged species. The type of chemical species used forrepulsion and the method of attachment to the channel walls can dependon the nature of the species being repelled and the nature of the wallsand the species being attached. Such surface modification techniques arewell known in the art. The walls may be functionalized before or afterthe device is assembled.

III. CAR T Cells

Methods for making and using CAR T cells are well known in the art.Procedures have been described in, for example, U.S. Pat. Nos.9,629,877; 9,328,156; 8,906,682; US 2017/0224789; US 2017/0166866; US2017/0137515; US 2016/0361360; US 2016/0081314; US 2015/0299317; and US2015/0024482; each of which is incorporated by reference herein in itsentirety.

IV. Separation Processes that Use DLD

The DLD devices described herein can be used to purify cells, cellularfragments, cell adducts, or nucleic acids. As discussed herein, thesedevices can also be used to separate a cell population of interest froma plurality of other cells. Separation and purification of bloodcomponents using devices can be found, for example, in US PublicationNo. US2016/0139012, the teaching of which is incorporated by referenceherein in its entirety. A brief discussion of a few illustrativeseparations is provided below.

A. Viable Cells

In one embodiment devices are used in procedures designed to separate aviable cell from a nonviable cell. The term “viable cell” refers to acell that is capable of growth, is actively dividing, is capable ofreproduction, or the like. In instances where a viable cell has a sizethat is greater than a nonviable cell, DLD devices can be designed tocomprise a critical size that is greater than a predetermined size ofthe nonviable cell and less than a predetermined size of the viablecell. The critical size may be as little as 1.1 fold greater than (orless than) the predetermined size of the nonviable cell but generally,larger degrees (or smaller) are preferred, e.g., about 1.2 fold-2 fold,and preferably 3-10 fold.

B. Adherent Cells

In another embodiment, DLD devices can be used to in procedures toseparate adherent cells. The term “adherent cell” as used herein refersto a cell capable of adhering to a surface. Adherent cells includeimmortalized cells used in cell culturing and can be derived frommammalian hosts. In some instances, the adherent cell may be trypsinizedprior to purification. Examples of adherent cells include MRC-5 cells;HeLa cells; Vero cells; NIH 3T3 cells; L929 cells; Sf21 cells; Sf9cells; A549 cells; A9 cells; AtT-20 cells; BALB/3T3 cells; BHK-21 cells;BHL-100 cells; BT cells; Caco-2 cells; Chang cells; Clone 9 cells; CloneM-3 cells; COS-1 cells; COS-3 cells; COS-7 cells; CRFK cells; CV-1cells; D-17 cells; Daudi cells; GH1 cells; GH3 cells; HaK cells; HCT-15cells; HL-60 cells; HT-1080 cells; HT-29 cells; HUVEC cells; I-10 cells;IM-9 cells; JEG-2 cells; Jensen cells; Jurkat cells; K-562 cells; KBcells; KG-1 cells; L2 cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells;WI-38 cells; WISH cells; XC cells; Y-1 cells; CHO cells; Raw 264.7;BHK-21 cells; HEK 293 cells to include 293A, 293T and the like; HEP G2cells; BAE-1 cells; SH-SY5Y cells; and any derivative thereof to includeengineered and recombinant strains.

In some embodiments, procedures may involve separating cells from adiluent such as growth media, which may provide for the efficientmaintenance of a culture of the adherent cells. For example, a cultureof adherent cells in a growth medium can be exchanged into atransfection media comprising transfection reagents, into a secondgrowth medium designed to elicit change within the adherent cell such asdifferentiation of a stem cell, or into sequential wash buffers designedto remove compounds from the culture.

In a particularly preferred procedure, adherent cells are purifiedthrough association with one or more carriers that bind in a way thatpromotes DLD separation. The carriers may be of the type describedherein and binding may stabilize and/or activate the cells. A carrierwill typically be in the rage of 1-1000 μm but may sometimes also beoutside of this range.

The association between a carrier and a cell should produce a complex ofincreased size relative to other material not associated with thecarrier. Depending of the particular size of the cells and carriers andthe number of cells and carriers present, a complex may be anywhere froma few percent larger than the uncomplexed cell to many times the size ofthe uncomplexed cell. In order to facilitate separations, an increase ofat least 20% is desirable with higher percentages (50; 100; 1000 ormore) being preferred.

C. Activated Cells

The DLD devices can also be used in procedures for separating anactivated cell or a cell capable of activation, from a plurality ofother cells. The cells undergoing activation may be grown on a largescale but, in a preferred embodiment, the cells are derived from asingle patient and DLD is performed within at least few hours aftercollection. The terms “activated cell” or “cell capable of activation”refers to a cell that has been, or can be activated, respectively,through association, incubation, or contact with a cell activator.Examples of cells capable of activation can include cells that play arole in the immune or inflammatory response such as: T cells, B cells;regulatory T cells, macrophages, dendritic cells, granulocytes, innatelymphoid cells, megakaryocytes, natural killer cells, thrombocytes,synoviocytes, and the like; cells that play a role in metabolism, suchas beta cells, liver cells, and pancreatic cells; and recombinant cellscapable of inducible protein expression such as DE3 lysogenized E. colicells, yeast cells, plant cells, etc.

Typically, one or more carriers will have the activator on theirsurface. Examples of cell activators include proteins, antibodies,cytokines, CD3, CD28, antigens against a specific protein, helper Tcells, receptors, and glycoproteins; hormones such as insulin, glucagonand the like; IPTG, lactose, allolactose, lipids, glycosides, terpenes,steroids, and alkaloids. The activatable cell should be at leastpartially associated with carriers through interaction between theactivatable cell and cell activator on the surface of the carriers. Thecomplexes formed may be just few percent larger than the uncomplexedcell or many times the size of the uncomplexed cell. In order tofacilitate separations, an increase of at least 20% is desirable withhigher percentages (40, 50 100 1000 or more) being preferred.

D. Separating Cells from Toxic Material

DLD can also be used in purifications designed to remove compounds thatmay be toxic to a cell or to keep the cells free from contamination by atoxic compound. Examples include an antibiotic, a cryopreservative, anantifungal, a toxic metabolite, sodium azide, a metal ion, a metal ionchelator, an endotoxin, a plasticizer, a pesticide, and any combinationthereof. The device can be used to remove toxic compounds from cells toensure consistent production of material from the cells. In someinstances, the cell can be a log phase cell. The term “log phase cell”refers to an actively dividing cell at a stage of growth characterizedby exponential logarithmic growth. In log phase, a cell population candouble at a constant rate such that plotting the natural logarithm ofcell number against time produces a straight line.

The ability to separate toxic material may be important for a widevariety of cells including: bacterial strains such as BL21, Tuner,Origami, Origami B, Rosetta, C41, C43, DH5α, DH10β, or XL1Blue; yeaststrains such as those of genera Saccharomyces, Pichia, Kluyveromyces,Hansenula and Yarrowia; algae; and mammalian cell cultures, includingcultures of MRC-5 cells; HeLa cells; Vero cells; NIH 3T3 cells; L929cells; Sf21 cells; Sf9 cells; A549 cells; A9 cells; AtT-20 cells;BALB/3T3 cells; BHK-21 cells; BHL-100 cells; BT cells; Caco-2 cells;Chang cells; Clone 9 cells; Clone M-3 cells; COS-1 cells; COS-3 cells;COS-7 cells; CRFK cells; CV-1 cells; D-17 cells; Daudi cells; GH1 cells;GH3 cells; HaK cells; HCT-15 cells; HL-60 cells; HT-1080 cells; HT-29cells; HUVEC cells; I-10 cells; IM-9 cells; JEG-2 cells; Jensen cells;Jurkat cells; K-562 cells; KB cells; KG-1 cells; L2 cells; LLC-WRC 256cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells; XC cells; Y-1cells; CHO cells; Raw 264.7; BHK-21 cells; HEK 293 cells to include293A, 293T and the like; HEP G2 cells; BAE-1 cells; SH-SY5Y cells; stemcells and any derivative thereof to include engineered and recombinantstrains.

E. Purification of Material Secreted from Cells

The DLD devices may also be used in the purification of materialsecreted from a cell. Examples of such secreted materials includesproteins, peptides, enzymes, antibodies, fuel, biofuels such as thosederived from algae, polymers, small molecules such as simple organicmolecules, complex organic molecules, drugs and pro-drugs, carbohydratesand any combination thereof. Secreted products can includetherapeutically useful proteins such as insulin, Imatinib, T cells, Tcell receptors, Fc fusion proteins, anticoagulants, blood factors, bonemorphogenetic proteins, engineered protein scaffolds, enzymes, growthfactors, hormones, interferons, interleukins, and thrombolytics.

FIG. 15 is a schematic depicting the use of DLD in the purification ofsecreted products. In some instances, the cells may be in an aqueoussuspension of buffer, growth medium, or the like, such that the cellsecretes product into the suspension. Examples of such secreted productsinclude proteins, peptides, enzymes, antibodies, fuel, biofuels such asthose derived from algae, polymers, small molecules such as simpleorganic molecules, complex organic molecules, drugs and pro-drugs,carbohydrates and any combination thereof. Secreted products can includetherapeutically useful proteins such as insulin, Imatinib, T cells, Tcell receptors, Fc fusion proteins, anticoagulants, blood factors, bonemorphogenetic proteins, engineered protein scaffolds, enzymes, growthfactors, hormones, interferons, interleukins, and thrombolytics.

Purification might be carried out, for example, in situations wherecells have a predetermined size that is greater than a predeterminedsize of the secreted compound, where the predetermined size of the cellis greater than or equal to a critical size, and the predetermined sizeof the secreted compound is less than the critical size. In such aconfiguration, when applied to a DLD device, the cells can be deflectedin a first direction while the secreted compound can be deflected in asecond direction, thereby separating the secreted compound from thecell. Also, a secreted protein may be captured by a large carrier thatbinds in a way that promotes DLD separation. DLD may then be performedand the carrier-protein complex may then be treated to further purify,or release, the protein.

Such processes can be carried out in an iterative fashion such that apopulation of separated particles can be continuously looped back into adevice for further separation. In this regard, FIGS. 16 and 17 areschematics of an iterative process in which separated cells are loopedback into the DLD device after separation. In some instances, the cellsmay be looped from a first device into a second, different device withobstacles comprising different critical sizes. Such a system can allowsystematic separation of a plurality of size ranges by manipulating therange of critical sizes. In other instances, cells may be looped back tothe same device used previously to separate the isolated particles. Thissystem can be advantageous for continuous purification of activelydividing cells or compounds being actively expressed. For example, sucha method could be combined with the method of purifying the secretedproduct to both collect the secreted product from one flow stream andthe cell producing the secreted product from another flow stream.Because the cells can continuously produce the secreted product, thepurified cells can be reapplied to the device to continuously collectthe secreted product from the cells.

F. Purity and Yields

The purity, yields and viability of cells produced by the DLD methodsdiscussed herein will vary based on a number of factors including thenature of the starting material, the exact procedure employed and thecharacteristics of the DLD device. Preferably, purifications, yields andviabilities of at least 60% should be obtained with, higher percentages,at least 70, 80 or 90% being more preferred. In a preferred embodiment,methods may be used to isolate leukocytes from whole blood, apheresisproducts or leukapheresis products with at least 70% purity, yield andviability with higher percentages (at least 80%, 85%, or 90%) beingpreferred.

V. Technological Background

Without being held to any particular theory, a general discussion ofsome technical aspects of microfluidics may help in understandingfactors that affect separations carried out in this field. A variety ofmicrofabricated sieving matrices have been disclosed for separatingparticles (Chou, et. al., Proc. Natl. Acad. Sci. 96:13762 (1999); Han,et al., Science 288:1026 (2000); Huang, et al., Nat. Biotechnol. 20:1048(2002); Turner et al., Phys. Rev. Lett. 88(12):128103 (2002); Huang, etal., Phys. Rev. Lett. 89:178301 (2002); U.S. Pat. Nos. 5,427,663;7,150,812; 6,881,317). Bump array (also known as “obstacle array”)devices have been described, and their basic operation is explained, forexample in U.S. Pat. No. 7,150,812, which is incorporated herein byreference in its entirety. A bump array operates essentially bysegregating particles passing through an array (generally, aperiodically-ordered array) of obstacles, with segregation occurringbetween particles that follow an “array direction” that is offset fromthe direction of bulk fluid flow or from the direction of an appliedfield (U.S. Pat. No. 7,150,812).

A. Bump Arrays

In some arrays, the geometry of adjacent obstacles is such that theportions of the obstacles defining the gap are symmetrical about theaxis of the gap that extends in the direction of bulk fluid flow. Thevelocity or volumetric profile of fluid flow through such gaps isapproximately parabolic across the gap, with fluid velocity and fluxbeing zero at the surface of each obstacle defining the gap (assumingno-slip flow conditions) and reaching a maximum value at the centerpoint of the gap. The profile being parabolic, a fluid layer of a givenwidth adjacent to one of the obstacles defining the gap contains anequal proportion of fluid flux as a fluid layer of the same widthadjacent to the other obstacle that defines the gap, meaning that thecritical size of particles that are ‘bumped’ during passage through thegap is equal regardless of which obstacle the particle travels near.

In some cases, particle size-segregating performance of an obstaclearray can be improved by shaping and disposing the obstacles such thatthe portions of adjacent obstacles that deflect fluid flow into a gapbetween obstacles are not symmetrical about the axis of the gap thatextends in the direction of bulk fluid flow. Such lack of flow symmetryinto the gap can lead to a non-symmetrical fluid flow profile within thegap. Concentration of fluid flow toward one side of a gap (i.e., aconsequence of the non-symmetrical fluid flow profile through the gap)can reduce the critical size of particles that are induced to travel inthe array direction, rather than in the direction of bulk fluid flow.This is because the non-symmetry of the flow profile causes differencesbetween the width of the flow layer adjacent to one obstacle thatcontains a selected proportion of fluid flux through the gap and thewidth of the flow layer that contains the same proportion of fluid fluxand that is adjacent to the other obstacle that defines the gap. Thedifferent widths of the fluid layers adjacent to obstacles define a gapthat exhibits two different critical particle sizes. A particletraversing the gap can be bumped (i.e., travel in the array direction,rather than the bulk fluid flow direction) if it exceeds the criticalsize of the fluid layer in which it is carried. Thus, it is possible fora particle traversing a gap having a non-symmetrical flow profile to bebumped if the particle travels in the fluid layer adjacent to oneobstacle, but to be not-bumped if it travels in the fluid layer adjacentto the other obstacle defining the gap.

In another aspect, decreasing the roundness of edges of obstacles thatdefine gaps can improve the particle size-segregating performance of anobstacle array. By way of example, arrays of obstacles having atriangular cross-section with sharp vertices can exhibit a lowercritical particle size than do arrays of identically-sized and -spacedtriangular obstacles having rounded vertices.

Thus, by sharpening the edges of obstacles defining gaps in an obstaclearray, the critical size of particles deflected in the array directionunder the influence of bulk fluid flow can be decreased withoutnecessarily reducing the size of the obstacles. Conversely, obstacleshaving sharper edges can be spaced farther apart than, but still yieldparticle segregation properties equivalent to, identically-sizedobstacles having less sharp edges.

B. Fractionation Range

Objects separated by size on microfluidic include cells, biomolecules,inorganic beads, and other objects. Typical sizes fractionated rangefrom 100 nanometers to 50 micrometers. However, larger and smallerparticles may also sometimes be fractionated.

C. Volumes

Depending on design, a device or combination of devices might be used toprocess between about 10 μl to at least 500 μl of sample, between about500 μl and about 40 mL of sample, between about 500 μl and about 20 mLof sample, between about 20 mL of sample and about 200 mL of sample,between about 40 mL of sample and about 200 mL of sample, or at least200 mL of sample.

D. Channels

A device can comprise one or multiple channels with one or more inletsand one or more outlets. Inlets may be used for sample or crude (i.e.,unpurified) fluid compositions, for buffers or to introduce reagents.Outlets may be used for collecting product or may be used as an outletfor waste. Channels may be about 0.5 to 100 mm in width and about 2-200mm long but different widths and lengths are also possible. Depth may be1-1000 μm and there may be anywhere from 1 to 100 channels or morepresent. Volumes may vary over a very wide range from a few μl to manyml and devices may have a plurality of zones (stages, or sections) withdifferent configurations of obstacles.

E. Gap Size (Edge-to-Edge Distance Between Posts or Obstacles)

Gap size in an array of obstacles (edge-to-edge distance between postsor obstacles) can vary from about a few (e.g., 1-500) micrometers or bemore than a millimeter. Obstacles may, in some embodiments have adiameter of 1-3000 micrometers and may have a variety of shapes (round,triangular, teardrop shaped, diamond shaped, square, rectangular etc.).A first row of posts can be located close to (e.g. within 5 μm) theinlet or be more than 1 mm away.

F. Stackable Chips

A device can include a plurality of stackable chips. A device cancomprise about 1-50 chips. In some instances, a device may have aplurality of chips placed in series or in parallel or both.

VI. Inventive Concepts

The numbered paragraphs below present inventive concepts that are partof the present application. These concepts are expressed in the form ofexample paragraphs E1-E273.

-   E1. A method of engineering a population of target cells,    comprising:    -   a) isolating the target cells from a crude fluid composition        wherein the isolation procedure comprises performing        Deterministic Lateral Displacement (DLD) on a microfluidic        device, wherein said device comprises:        -   i) at least one channel extending from a sample inlet to one            or more fluid outlets, wherein the channel is bounded by a            first wall and a second wall opposite from the first wall;        -   ii) an array of obstacles arranged in rows in the channel,            each subsequent row of obstacles being shifted laterally            with respect to a previous row, and wherein said obstacles            are disposed in a manner such that, when said crude fluid            composition is applied to an inlet of the device and            fluidically passed through the channel, target cells flow to            one or more collection outlets where an enriched product is            collected and contaminant cells or particles that are of a            different size than the target cells flow to one or more            waste outlets that are separate from the collection outlets;    -   b) genetically engineering the target cells obtained from the        collection outlet(s) to have a desired phenotype.-   E2. The method of E1, wherein said genetic engineering comprises    transfecting or transducing the target cells and the genetically    engineered target cells are expanded by culturing them in vitro.-   E3. The method of E2, wherein the yield of target cells exhibiting    the desired phenotype is at least 10% greater than identical cells    isolated by Ficoll centrifugation and not subjected to DLD.-   E4. The method of E1, wherein the crude fluid composition is blood    or a composition that has been obtained by performing apheresis or    leukapheresis on blood.-   E5. The method of any one of E1-4, wherein the target cells are    leukocytes.-   E6. The method of any one of E1-4, wherein the target cells are    B-cells, T cells, NK-cells, monocytes or progenitor cells.-   E7. The method of any one of E1-4, wherein the target cell a    dendritic cell.-   E8. The method of any one of E1-7, wherein said crude fluid    composition is obtained from a patient.-   E9. The method of E8, wherein, target cells in the crude fluid    composition are not bound to a carrier before being transduced or    transfected.-   E10. The method of E8, wherein target cells are bound to one or more    carriers in a way that promotes or complements DLD separation before    performing DLD.-   E11. The method of E9, wherein target cells are bound to one or more    carriers in a way that promotes or complements DLD separation after    performing DLD and either before or after transducing or    transfecting them.-   E12. The method of E10 or E11, wherein said one or more carriers    comprise on their surface an affinity agent that binds specifically    to said target cells.-   E13. The method of E12, wherein said agent is an antibody, an    activator, a hapten or an aptamer.-   E14. The method of any one of E10-13, wherein the diameter of said    carriers is at least as large as that of the target cells.-   E15. The method of any one of E10-13, wherein the diameters of all    of said carriers are no more than 50% as large as that of the target    cells.-   E16. The method of any one of E10-13, wherein the diameters of all    of said carriers are at least two times larger than that of the    target cells.-   E17. The method of any one of E10-13, wherein the diameters of all    of said carriers are no more than 25% as large as that of the target    cells.-   E18. The method of any one of E10-13, wherein one group of carriers    has a diameter at least as large as the target cells and a second    group of carriers has a diameter no more than 50% as large as that    of the target cells.-   E19. The method of any one of E10-13, wherein one group of carriers    has a diameter at least twice as large as the target cells and a    second group of carriers has a diameter no more than 25% as large as    the target cells.-   E20. The method of any one of E10-19, wherein said carriers are made    of collagen or a polysaccharide.-   E21. The method of anyone of E10-20, wherein said carriers are made    of gelatin or alginate.-   E22. The method of any one of E10-21, wherein the crude fluid    composition is obtained from a patient and no more than four hours    elapse from the time that the obtaining of the crude fluid    composition is complete until the target cells are first bound to a    carrier.-   E23. The method of any one of E10-21, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of a patient and no more than four hours elapse from the    time that apheresis or leukapheresis is completed until the target    cells are first bound to a carrier.-   E24. The method of any one of E1-23, wherein the crude fluid    composition is obtained from a patient and no more than five hours    elapse from the time that the obtaining of the crude fluid    composition is complete until the first time that target cells are    transfected or transduced.-   E25. The method of any one of E1-23, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of a patient and no more than five hours elapse from the    time that apheresis or leukapheresis is completed until the first    time that target cells are transfected or transduced.-   E26. The method of either E24 or E25, wherein no more than four    hours elapse until the first time that target cells are transfected    or transduced.-   E27. A method of producing Chimeric Antigen Receptor (CAR) T cells,    comprising:    -   a) obtaining a crude fluid composition comprising T cells;    -   b) performing Deterministic Lateral Displacement (DLD) on the        crude fluid composition using a microfluidic device comprising:        -   i) at least one channel extending from a sample inlet to one            or more fluid outlets, wherein the channel is bounded by a            first wall and a second wall opposite from the first wall;        -   ii) an array of obstacles arranged in rows in the channel,            each subsequent row of obstacles being shifted laterally            with respect to a previous row, and wherein said obstacles            are disposed in a manner such that, when the crude fluid            composition is applied to an inlet of the device and            fluidically passed through the channel, T cells in the            composition flow to one or more collection outlets where an            enriched product is collected, and cells, or particles that            are in the crude fluid composition and that are of a            different size than the T cells, flow to one or more waste            outlets that are separate from the collection outlets;    -   c) genetically engineering the T cells in the enriched product        obtained in step b) to produce the chimeric antigen receptors        (CARs) on their surface.-   E28. The method of E27, wherein said crude fluid composition is an    apheresis product or leukapheresis product obtained from blood from    a patient and wherein, when the crude fluid composition is applied    to an inlet of the device and fluidically passed through the    channel, T cells in the composition flow to one or more collection    outlets where an enriched product is collected, and red blood,    platelets or other particles that are in the crude fluid composition    and that are of a different size, flow to one or more waste outlets    that are separate from the collection outlets.-   E29. The method of either E27 or E28, wherein said genetic    engineering comprises transfecting or transducing the target cells    and the genetically engineered target cells are expanded further by    growing the cells in vitro.-   E30. The method of any one of E27-29, wherein the yield of T cells    expressing the chimeric receptors on their surface is at least 10%    greater than T cells isolated from the crude fluid composition by    Ficoll centrifugation and not subjected to DLD.-   E31. The method of E30, wherein the yield of T cells expressing the    chimeric receptors on their surface is at least 20% greater than T    cells isolated from the crude fluid composition by Ficoll    centrifugation and not subjected to DLD.-   E32. The method of E30, wherein the yield of T cells expressing the    chimeric receptors on their surface is at least 50% greater than T    cells isolated from the crude fluid composition by Ficoll    centrifugation and not subjected to DLD.-   E33. The method of any one of E27-32, wherein said CAR comprises a)    an extracellular region comprising antigen binding domain; b) a    transmembrane region; c) an intracellular region and wherein said    CAR T cells optionally comprise one or more recombinant sequences    that provide the cells with a molecular switch that, when triggered,    reduce CAR T cell number or activity.-   E34. The method of E33, wherein said antigen binding domain is a    single chain variable fragment (scFv), from antigen binding regions    of both heavy and light chains of a monoclonal antibody.-   E35. The method of E33 or E34 wherein said CAR comprises a hinge    region of 2-20 amino acids connecting the extracellular region and    the transmembrane region.-   E36. The method of E35, wherein said transmembrane region comprises    CD8 or CD28 protein sequences.-   E37. The method of any one of E33-36, wherein said intracellular    region comprises a signaling domain derived from CD3-zeta, CD137 or    a CD28 intracellular domain.-   E38. The method of any one of E27-37, wherein said crude fluid    composition comprising T cells is obtained from a patient with    cancer, an autoimmune disease or an infectious disease.-   E39. The method of E38 wherein, after obtaining the crude fluid    composition comprising T cells, the T cells in the fluid composition    are bound to one or more carriers in a way that promotes DLD    separation.-   E40. The method of E39, wherein T cells are bound to one or more    carriers in a way that promotes DLD separation before performing    DLD.-   E41. The method of E39, wherein T cells are bound to one or more    carriers in a way that promotes DLD separation after performing DLD    and either before or after they are genetically engineered.-   E42. The method of E39-41, wherein said one or more carriers    comprise on their surface an antibody or activator that binds    specifically to said T cells.-   E43. The method of any one of E39-42, wherein the diameters of all    of said carriers are at least as large as that of the T cells.-   E44. The method of any one of E39-42, wherein the diameters of all    of said carriers are no more than 50% as large as that of the T    cells.-   E45. The method of any one of E39-42, wherein the diameters of all    of said carriers are at least two times larger than that of the T    cells.-   E46. The method of any one of E39-42, wherein the diameters of all    of said carriers are no more than 25% as large as that of the T    cells.-   E47. The method of any one of E39-42, wherein one group of carriers    has a diameter at least as large as the T cells and a second group    of carriers has a diameter no more than 50% as large as that of the    T cells.-   E48. The method of any one of E39-42, wherein one group of carriers    has a diameter at least twice as large as the T cells and a second    group of carriers has a diameter no more than 25% as large as the T    cells.-   E49. The method of any one of E39-48, wherein said carriers are made    of collagen or a polysaccharide.-   E50. The method of any one of E39-49, wherein said carriers are made    of gelatin or alginate.-   E51. The method of any one of E39-50, wherein no more than four    hours elapse from the time that obtaining of the crude fluid    composition comprising T cells is completed until the T cells are    bound to a carrier.-   E52. The method of any one of E39-50, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of a patient and no more than four hours elapse from the    time that apheresis or leukapheresis is completed until the target    cells are bound to a carrier.-   E53. The method of any one of E27-50, wherein no more than five    hours elapse from the time that obtaining of the crude fluid    composition comprising T cells is completed until the first time    that T cells are transfected or transduced.-   E54. The method of any one of E27-50, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of a patient and no more than five hours elapse from the    time that apheresis or leukapheresis is completed until the first    time that T cells are transfected or transduced.-   E55. The method of either E53 or E54, wherein no more than four    hours elapse until the first time that T cells are transfected or    transduced.-   E56. The method of any one of E27-55 where all steps in producing    the CAR T cells are performed at the same facility where the a crude    fluid composition comprising T cells is obtained and all steps are    completed in a total of no more than four hours.-   E57. CAR T cells made by the method of any one of E27-55.-   E58. A method of treating a patient for cancer, an autoimmune    disease, or an infectious disease, comprising administering to said    patient CAR T cells engineered to express chimeric antigen receptors    that recognize antigens on cancer cells, autoimmune cells or    infectious cells from said patient, wherein said CAR T cells have    been produced by a process comprising:    -   a) obtaining a crude fluid composition comprising T cells from a        patient;    -   b) performing Deterministic Lateral Displacement (DLD) on the        crude fluid composition using a microfluidic device comprising:        -   i) at least one channel extending from a sample inlet to one            or more fluid outlets, wherein the channel is bounded by a            first wall and a second wall opposite from the first wall;        -   ii) an array of obstacles arranged in rows in the channel,            each subsequent row of obstacles being shifted laterally            with respect to a previous row, and wherein said obstacles            are disposed in a manner such that, when the crude fluid            composition is applied to an inlet of the device and            fluidically passed through the channel, T cells in the            composition flow to one or more collection outlets where an            enriched product is collected, and cells, or particles that            are in the crude fluid composition and that are of a            different size than the T cells, flow to one more or waste            outlets that are separate from the collection outlets;    -   c) genetically engineering the T cells obtained in step b) to        express chimeric antigen receptors (CARs) on their surface;    -   d) expanding the number of engineered T cells by growing the        cells in vitro; and    -   e) administering the engineered T cells to the patient from        which the crude fluid composition was obtained.-   E59. The method of E58, wherein said crude fluid composition is an    apheresis product or leukapheresis product obtained from blood from    said patient and wherein, when the crude fluid composition is    applied to an inlet of the device and fluidically passed through the    channel, T cells in the composition flow to one or more collection    outlets where an enriched product is collected, and red blood cells,    platelets or other particles that are in the crude fluid composition    and that are of a different size, flow to one more waste outlets    that are separate from the collection outlets.-   E60. The method of E58 or E59, wherein genetic engineering comprises    transfecting or transducing the target cells.-   E61. The method of E60, wherein the yield of cells expressing the    chimeric receptors on their surface is at least 10% greater than T    cells isolated from the crude fluid composition by Ficoll    centrifugation and not subjected to DLD.-   E62. The method of E60, wherein the yield of target cells expressing    the chimeric receptors on their surface is at least 50% greater than    T cells isolated from the crude fluid composition by Ficoll    centrifugation and not subjected to DLD.-   E63. The method of any one of E58-62, wherein said CAR comprises a)    an extracellular region comprising antigen binding domain; b) a    transmembrane region; and c) an intracellular region and wherein    said CAR T cells optionally comprise one or more recombinant    sequences that provide the cells with a molecular switch that, when    triggered, reduce CAR T cell number or activity.-   E64. The method of E63, wherein said antigen binding domain is a    single chain variable fragment (scFv), from the antigen binding    regions of both heavy and light chains of a monoclonal antibody.-   E65. The method of E63 or E64 wherein said CAR comprises a hinge    region of 2-20 amino acids connecting the extracellular region and    the transmembrane region.-   E66. The method of any one of E63-65, wherein said transmembrane    region comprises CD8 or CD28 protein sequences.-   E67. The method of any one of E63-66, wherein said intracellular    region comprises a signaling domain derived from CD3-zeta, CD137, a    CD28 intracellular domain.-   E68. The method of any one of E58-67, wherein said patient has    leukemia.-   E69. The method of E68, wherein said leukemia is acute lymphoblastic    leukemia.-   E70. The method of E68 or E69, wherein said CAR recognizes as an    antigen CD19 or CD20.-   E71. The method of any one of E58-67, wherein said patient has a    solid tumor.-   E72. The method of E71, wherein said CAR recognizes an antigen    selected from the group consisting of: CD22; RORI; mesothelin;    CD33/IL3Ra; c-Met; PSMA; Glycolipid F77; EGFRvIII; GD-2; NY-ESO-1    TCR; MAGE A3 TCR; and combinations thereof.-   E73. The method of any one of E58-72 wherein, after obtaining the    crude fluid composition comprising T cells, the T cells in the fluid    are bound to a carrier in a way that promotes DLD separation.-   E74. The method of E73, wherein T cells are bound to one or more    carriers in a way that promotes DLD separation before performing    DLD.-   E75. The method of E73, wherein T cells are bound to one or more    carriers in a way that promotes DLD separation after performing DLD    and either before or after the T cells are genetically engineered to    express chimeric receptors.-   E76. The method of E73-75, wherein said one or more carriers    comprise on their surface an antibody or activator that binds    specifically to said T cells.-   E77. The method of any one of E73-76, wherein the diameters of all    of said carriers are at least as large as that of the T cells.-   E78. The method of any one of E73-76, wherein the diameters of all    of said carriers are no more than 50% as large as that of the T    cells.-   E79. The method of any one of E73-76, wherein the diameters of all    of said carriers are at least two times larger than that of the T    cells.-   E80. The method of any one of E73-76, wherein the diameters of all    of said carriers are no more than 25% as large as that of the T    cells.-   E81. The method of E80, wherein one group of carriers has a diameter    at least as large as the T cells and a second group of carriers has    a diameter no more than 50% as large as that of the T cells.-   E82. The method of any one of E73-81, wherein one group of carriers    has a diameter at least twice as large as the T cells and a second    group of carriers has a diameter no more than 25% as large as the T    cells.-   E83. The method of any one of E73-82, wherein said carriers are made    of collagen or a polysaccharide.-   E84. The method of any one of E73-83, wherein said carriers are made    of gelatin or alginate.-   E85. The method of any one of E73-84, wherein no more than four    hours elapse from the time that obtaining of the crude fluid    composition comprising T cells is completed until the T cells are    bound to a carrier.-   E86. The method of any one of E73-84, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of a patient and no more than four hours elapse from the    time that apheresis or leukapheresis is completed until the target    cells are bound to a carrier.-   E87. The method of any one of E73-84, wherein no more than five    hours elapse from the time that obtaining of the crude fluid    composition comprising T cells is completed until the first time    that the T cells are transfected or transduced.-   E88. The method of any one of E73-84, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of a patient and no more than five hours elapse from the    time that apheresis or leukapheresis is completed until the first    time that T cells are transfected or transduced.-   E89. The method of E87 or E88, wherein no more than four hours    elapse until the first time that T cells are transfected or    transduced.-   E90. The method of any one of E58-89, wherein T cells are available    for administration to a patient at least 1 day earlier than for    cells processed via a method not including DLD.-   E91. The method of any one of E58-89, wherein target cells are    available for administration to a patient at least 3 days earlier    than for cells processed via a method not including DLD.-   E92. A method of collecting target cells from a patient comprising:    -   a) obtaining from the patient a crude fluid composition        comprising target cells;    -   b) performing Deterministic Lateral Displacement (DLD) on the        crude fluid composition comprising target cells using a        microfluidic device to obtain a composition enriched in target        cells;    -   wherein, either before or after DLD, target cells are bound to        one or more carriers in a way that promotes DLD separation and        wherein no more than five hours elapse from the time that the        obtaining of the crude fluid composition comprising target cells        from the patient is completed until the target cells are bound        to a carrier.-   E93. The method of E92, wherein said one or more carriers comprise    on their surface an antibody or activator that binds specifically to    said target cells.-   E94. The method of either E92 or E93, wherein the diameters of all    of said carriers are at least as large as that of the target cells.-   E95. The method of either E92 or E93, wherein the diameters of all    of said carriers are no more than 50% as large as that of the target    cells.-   E96. The method of either E92 or E93, wherein the diameters of all    of said carriers are at least two times larger than that of the    target cells.-   E97. The method of either E92 or E93, wherein the diameters of all    of said carriers are no more than 25% as large as that of the target    cells.-   E98. The method of either E92 or E93, wherein one group of carriers    has a diameter at least as large as the target cells and a second    group of carriers has a diameter no more than 50% as large as that    of the target cells.-   E99. The method of either E92 or E93, wherein one group of carriers    has a diameter at least twice as large as the T cells and a second    group of carriers has a diameter no more than 25% as large as the T    cells.-   E100. The method of any one of E92-99, wherein said carriers are    made of collagen or a polysaccharide.-   E101. The method of any one of E92-100, wherein said carriers are    made of gelatin or alginate.-   E102. The method of any one of E92-101, wherein no more than four    hours elapse from the time that the obtaining of the crude fluid    composition comprising target cells is completed until the target    cells are bound to a carrier.-   E103. The method of any one of E92-101, wherein no more than three    hours elapse from the time that the obtaining of the crude fluid    composition comprising target cells is completed until the target    cells are bound to a carrier.-   E104. The method of any one of E92-103, wherein said crude fluid    composition comprising target cells is obtained by performing    apheresis or leukapheresis on blood from the patient.-   E105. The method of any one of E92-104 wherein target cells in the    composition enriched in target cells by DLD are transduced using a    viral vector.-   E106. The method of E105 wherein target cells are transfected    electrically, chemically or by means of nanoparticles.-   E107. The method of anyone of E92-106, wherein said microfluidic    device comprises:    -   a) at least one channel extending from a sample inlet to one or        more fluid outlets, wherein the channel is bounded by a first        wall and a second wall opposite from the first wall;    -   b) an array of obstacles arranged in rows in the channel, each        subsequent row of obstacles being shifted laterally with respect        to a previous row, and wherein said obstacles are disposed in a        manner such that, when said crude fluid composition comprising        target cells is applied to an inlet of the device and        fluidically passed through the channel, target cells flow to one        or more collection outlets where an enriched product is        collected and contaminant cells, or particles that are in the        crude fluid composition and that are of a different size than        the target cells flow to one or more waste outlets that are        separate from the collection outlets.-   E108. The method of anyone of E92-107, wherein said target cells are    T cells.-   E109. The method of E108, wherein said T cells are selected from the    group consisting of: Natural Killer T cells; Central Memory T cells;    Helper T cells and Regulatory T cells.-   E110. The method of anyone of E92-107, wherein said target cells are    stem cells.-   E111. The method of any one of E92-107, wherein said target cells    are B cells, macrophages, dendritic cells, or granulocytes.-   E112. The method of any one of E92-111, wherein said crude fluid    composition comprising target cells comprises one or more additives    that act as anticoagulants or that prevent the activation of    platelets.-   E113. The method of E112, wherein said additives are selected from    the group consisting of ticlopidine, inosine, protoctechuic acid,    acetylsalicylic acid, and tirofiban.-   E114. The method of any one of E92-113, wherein steps a) and b) are    both carried out at the site where the crude fluid composition    comprising target cells is obtained from the patient.-   E115. The method of any one of E92-114, wherein no more than four    hours elapse from the time that the obtaining of the crude fluid    composition comprising target cells from the patient is completed    until the target cells are bound to a carrier.-   E116. The method of any one of E92-114, wherein the crude fluid    composition is an apheresis or leukapheresis product derived from    the blood of the patient and no more than four hours elapse from the    time that apheresis or leukapheresis is completed until the target    cells are bound to a carrier.-   E117. The method of any one of E92-116, wherein said method further    comprises:    -   c) genetically engineering and/or expanded cells in number;        and/or    -   d) treating the same patient from which the target cells were        obtained with the target cells collected.-   E118. The method of E117, wherein, after step d), said target cells    are cryopreserved.-   E119. The method of either E117 or E118, wherein target cells that    are cultured in step c) are T cells that are cultured in the    presence of an activator.-   E120. The method of E119, wherein the activator is bound to a    carrier.-   E121. The method of any one of E58-89, wherein target cells are    available for administration to the patient at least 1 day earlier    than for cells processed via a method not including DLD.-   E122. The method of any one of E58-89, wherein target cells are    available for administration to the patient at least 3 days earlier    than for cells processed via a method not including DLD.-   E123. Target cells produced by the method of any one of E92-122.-   E124. A method of treating a patient for a disease or condition    comprising administering to said patient the target cells of E123.-   E125. A method of separating an adherent cell from a plurality of    other cells comprising:    -   a) contacting a crude fluid composition comprising the plurality        of other cells and the adherent cell with one or more carriers        that bind in a way that promotes DLD separation, wherein the        adherent cell is at least partially associated with carriers        upon or after contact to generate a carrier associated adherent        cell complex, wherein the carrier associated adherent cell        complex comprises an increased size relative to cells in the        plurality of other cells, and wherein the size of the carrier        associated adherent cell complex is greater than or equal to a        critical size, and the cells in the plurality of other cells        comprise a size less than the critical size;    -   b) applying the crude fluid composition to a device, wherein the        device comprises an array of obstacles arranged in rows, wherein        the rows are shifted laterally with respect to one another,        wherein the rows are configured to deflect a particle greater        than or equal to the critical size in a first direction and a        particle less than the critical size in a second direction; and    -   c) flowing the sample comprising the carrier associated adherent        cell complex through the device, wherein the carrier associated        adherent cell complex is deflected by the obstacles in the first        direction, and the cells in the plurality of other cells are        deflected in the second direction, thereby separating the        carrier associated adherent cell complex from the other cells of        the plurality;    -   d) collecting a fluid composition comprising the separated        carrier associated adherent cell complex.-   E126. The method of E125, wherein said adherent cell is collected    from a patient as part of a crude fluid composition comprising said    adherent cell and a plurality of other cells, and wherein no more    than three hours elapse from the time that the obtaining of the    crude fluid composition from the patient is completed until the    adherent cell is bound to a carrier for the first time.-   E127. The method of E125, wherein no more than two hours elapse from    the time that the obtaining of the crude fluid composition from the    patient is completed until the adherent cell is bound to the carrier    for the first time.-   E128. The method of E125, wherein no more than one hour elapses from    the time that the obtaining of the crude fluid composition from the    patient is completed until the adherent cell is bound to the carrier    for the first time.-   E129. The method of E125, wherein no more than four hours elapse    from the time that the obtaining of the crude fluid composition from    the patient is completed until the adherent cell or the carrier    adherent cell complex is collected from the device for the first    time.-   E130. The method of E125, wherein no more than four hours elapse    from the time that the obtaining of the crude fluid composition from    the patient is completed until the adherent cell or the carrier    adherent cell complex is collected from the device for the first    time.-   E131 The method of any one of E125-130, wherein said carrier    comprises on its surface an antibody or activator that binds    specifically to said adherent cell.-   E132. The method of any one of E125-131, wherein the diameter of    said carrier is at least as large as that of the adherent cell.-   E133. The method of any one of E125-131, wherein the diameters of    all of said carriers are at least twice as large as that of the    adherent cell.-   E134. The method of any one of E125-131, wherein the diameters of    all of said carriers are at least ten times as large as that of the    adherent cell.-   E135. The method of any one of E125-131, wherein the diameters of    all of said carriers are 10-600 μm.-   E136. The method of any one of E125-135, wherein the adherent cell    is selected from the group consisting of: an MRC-5 cell; a HeLa    cell; a Vero cell; an NIH 3T3 cell; an L929 cell; a Sf21 cell; a Sf9    cell; an A549 cell; an A9 cell; an AtT-20 cell; a BALB/3T3 cell; a    BHK-21 cell; a BHL-100 cell; a BT cell; a Caco-2 cell; a Chang cell;    a Clone 9 cell; a Clone M-3 cell; a COS-1 cell; a COS-3 cell; a    COS-7 cell; a CRFK cell; a CV-1 cell; a D-17 cell; a Daudi cell; a    GH1 cell; a GH3 cell; an HaK cell; an HCT-15 cell; an HL-60 cell; an    HT-1080 cell; a HEK cell, HT-29 cell; an HUVEC cell; an I-10 cell;    an IM-9 cell; a JEG-2 cell; a Jensen cell; a Jurkat cell; a K-562    cell; a KB cell; a KG-1 cell; an L2 cell; an LLC-WRC 256 cell; a    McCoy cell; a MCF7 cell; a WI-38 cell; a WISH cell; an XC cell; a    Y-1 cell; a CHO cell; a Raw 264.7 cell; a HEP G2 cell; a BAE-1 cell;    an SH-SY5Y cell, and any derivative thereof.-   E137. The method of anyone of E125-135, wherein the adherent cell is    a stem cell.-   E138. A method of separating an activated cell from a plurality of    other cells comprising:    -   a) contacting a crude fluid composition comprising a cell        capable of activation and the plurality of other cells with one        or more carriers, wherein at least one carrier comprises a cell        activator, wherein the cell activator is at least partially        associated with the cell capable of activation by the cell        activator upon or after contact to generate a carrier associated        cell complex, wherein the association of the cell activator with        the cell capable of activation by the cell activator at least        partially activates the cell capable of activation, wherein the        carrier associated cell complex comprises an increased size        relative to cells in the plurality of other cells, and wherein a        size of the carrier associated cell complex is greater than or        equal to a critical size, and the cells in the plurality of        other cells comprise a size less than the critical size;    -   b) applying the sample to a device, wherein the device comprises        an array of obstacles arranged in rows; wherein the rows are        shifted laterally with respect to one another, wherein the rows        are configured to deflect a particle greater than or equal to        the critical size in a first direction and a particle less than        the critical size in a second direction; and    -   c) flowing the sample through the device, wherein the carrier        associated cell complex is deflected by the obstacles in the        first direction, and the cells in the plurality of other cells        are deflected in the second direction, thereby separating the        activated cell from the other cells of the plurality;    -   d) collecting a fluid composition comprising the separated        carrier associated cell complex.-   E139. The method of E138, wherein the cell capable of activation is    selected from the group consisting of: a T cell, a B cell, a    regulatory T cell, a macrophage, a dendritic cell, a granulocyte, an    innate lymphoid cell, a megakaryocyte, a natural killer cell, a    thrombocyte, a synoviocyte, a beta cell, a liver cell, a pancreatic    cell; a DE3 lysogenized cell, a yeast cell, a plant cell, and a stem    cell.-   E140. The method of E138 or E139, wherein the cell activator is a    protein.-   E141. The method of E140, wherein the protein is an antibody.-   E142. The method of E140, wherein the protein is selected from the    group consisting of: CD3, CD28, an antigen, a helper T cell, a    receptor, a cytokine, a glycoprotein, and any combination thereof.-   E143. The method of E138, wherein the cell activator is selected    from the group consisting of insulin, IPTG, lactose, allolactose, a    lipid, a glycoside, a terpene, a steroid, an alkaloid, and any    combination thereof.-   E144. The method of any one of E138-143, wherein said cell capable    of activation is collected from a patient as part of a crude fluid    composition comprising said cell capable of activation and a    plurality of other cells, and wherein no more than four hours elapse    from the time that the obtaining of the crude fluid composition from    the patient is completed until the cell capable of activation is    bound to the carrier.-   E145. The method of anyone of E138-143, wherein no more than three    hours elapse from the time that the obtaining of the crude fluid    composition from the patient is completed until the cell capable of    activation is bound to the carrier.-   E146. The method of E138-143, wherein no more than two hours elapse    from the time that the obtaining of the crude fluid composition from    the patient is completed until the cell capable of activation is    bound to the carrier.-   E147. The method of any one of E138-143, wherein no more than four    hours elapse from the time that the obtaining of the crude fluid    composition from the patient is completed until step c) is    completed.-   E148. The method of any one of E138-143, wherein no more than three    hours elapse from the time that the obtaining of the crude fluid    composition from the patient is completed until step c) is    completed.-   E149. The method of any one of E138-148, wherein the diameters of    all of said carriers are at least as large as the cell capable of    activation.-   E150. The method of any one of E138-148, wherein the diameters of    all of said carriers are at least twice as large as that of the cell    capable of activation.-   E151. The method of any one of E138-148, wherein the diameters of    all of said carriers are at least ten times as large as that of the    cell capable of activation.-   E152. The method of any one of E138-148, wherein the diameters of    said carriers are 10-600 μm.-   E153. A method of continuously purifying a secreted product from a    cell comprising:    -   a) obtaining a fluid composition comprising the cell, wherein        the cell is suspended in the fluid composition, wherein the cell        secretes the secreted product into the suspension, wherein the        cell has a predetermined size that is greater than a        predetermined size of the secreted product, and wherein the        predetermined size of the cell is greater than or equal to a        critical size, and the predetermined size of the secreted        product is less than the critical size;    -   b) applying the fluid composition comprising the cell to a        device, wherein the device comprises an array of obstacles        arranged in rows; wherein the rows are shifted laterally with        respect to one another, wherein the rows are configured to        deflect a particle greater than or equal to the critical size in        a first direction and a particle less than the critical size in        a second direction;    -   c) flowing the sample through the device, wherein the cell is        deflected by the obstacles in the first direction, and the        secreted product is deflected in the second direction, thereby        separating the secreted product from the cell;    -   d) collecting the secreted product, thereby producing a sample        of the secreted product that is substantially pure;    -   e) collecting a recovered fluid composition comprising the        separated cell; and    -   f) re-applying the recovered fluid composition comprising the        separated cell to the device and repeating steps (a) through        (e); thereby continuously purifying the secreted product from        the cell.-   E154. The method of E153, wherein the secreted product is selected    from the group consisting of: a protein, an antibody, a biofuel, a    polymer, a small molecule, and any combination thereof.-   E155. The method of E153, wherein the cell is selected from the    group consisting of: a bacterial cell, an algae cell, a mammalian    cell, and a tumor cell.-   E156. A method for decreasing the ratio of platelets to leukocytes    in an apheresis sample, comprising performing deterministic lateral    displacement (DLD) on the sample, in the absence of centrifugation    or elutriation, wherein a product is obtained in which the ratio of    platelets to leukocytes is at least 20% lower than the ratio    obtained with the same procedure performed using centrifugation or    elutriation instead of DLD.-   E157. The method of E156, wherein a product is obtained in which the    ratio of platelets to leukocytes is at least 20% lower than the    ratio obtained with the same procedure performed using density    gradient centrifugation or counterflow centrifugation.-   E158. The method of E156, wherein a product is obtained in which the    ratio of platelets to leukocytes is at least 20% lower than the    ratio obtained with the same procedure performed using elutriation.-   E159. The method of E156, wherein a product is obtained in which the    ratio of platelets to leukocytes is at least 50% lower than the    ratio obtained using centrifugation or elutriation instead of DLD.-   E160. The method of any one of E156-159, wherein there are no    separation steps performed on the apheresis sample prior to DLD.-   E161. The method of any one of E156-160, wherein DLD is performed in    a buffer that does not comprise intercalators that alter the size of    platelets and that does not promote platelet aggregation.-   E162. The method of any one of E156-159 wherein DLD is performed in    a buffer that does not comprise dextran or other highly charge    polymers.-   E163. The method of any one of E156-162, wherein the total number of    platelets in the product is at least 70% lower than in the apheresis    sample.-   E164. The method of any one of E156-163, wherein the total number of    platelets in the product is at least 90% lower than in the apheresis    sample.-   E165. A method for purifying T cells from an apheresis sample,    comprising performing DLD on the sample, followed by an affinity    separation step and expansion of the T cells by culturing in the    presence of activator, wherein the number of T cells obtained is at    least twice as high as the number produced by the same procedure    performed using Ficoll centrifugation instead of DLD.-   E166. The method of E165, wherein the affinity separation step    comprises the use of magnetic beads or particles comprising an    antibody binding to CD3.-   E167. The method of E165, wherein the number of T cells obtained    after 14 days in culture is at least two times higher than the    number produced by the same procedure performed using Ficoll    centrifugation instead of DLD.-   E168. The method of E165, wherein the number of T cells obtained    after 14 days in culture is at least four times higher than the    number produced by the same procedure performed using Ficoll    centrifugation instead of DLD.-   E169. The method of any one of E156-168 wherein, when cells in the    DLD product are transformed with a vector to express a recombinant    phenotype, the yield of cells exhibiting the desired phenotype is at    least 10% greater than for identical cells isolated by Ficoll    centrifugation and not subjected to DLD.-   E170. The method of any one of E156-169, wherein when cells in the    DLD product are transformed with a vector to express a recombinant    phenotype, the yield of T cells exhibiting the desired phenotype is    at least 20% greater than for identical cells isolated by Ficoll    centrifugation and not subjected to DLD.-   E171. The method of any one of E163-165 wherein the percentage of    memory T cells in the DLD product relative to the total number of T    cells is at least 10% higher than the percentage produced using the    same procedure but with Ficoll centrifugation instead of DLD.-   E172. The method of any one of E156-171, wherein the method is used    to produce T cells for CAR T cell therapy and the time needed to    produce a sufficient number of cells to treat a patient is reduced    by at least 20% using DLD instead of Ficoll centrifugation.-   E173. The method of E172, wherein the process for producing CART    cells does not include a step in which cells are frozen.-   E174. The method of either E172 or E173, wherein the processing of T    cells is performed at the same site where apheresis is performed.-   E175. The method of any one of E172-174, wherein T cells are    genetically transformed at the same site where apheresis is    performed.-   E176. The method of any one of E156-169, wherein no more than one    hour elapses from the time that the apheresis sample collection is    completed until the time that DLD is performed.-   E177. A method for decreasing the ratio of platelets to leukocytes    in an apheresis sample, comprising performing deterministic lateral    displacement (DLD) on the sample, in the absence of centrifugation    or elutriation, wherein a product is obtained in which the total    number of platelets in the product is at least 90% lower than in the    apheresis sample.-   E178. The method of E177, wherein DLD is performed in a buffer that    does not comprise intercalators that alter the size of platelets and    that does not promote platelet aggregation.-   E179. The method of E177, wherein DLD is performed in a buffer that    does not comprise dextran or other highly charge polymers.-   E180. A method of producing CART cells, comprising:    -   a) obtaining a sample composition from a patient by apheresis,        wherein said sample composition comprises T cells;    -   b) performing DLD on the sample composition to reduce the total        number of platelets present by at least 70%, wherein DLD is        carried out on a microfluidic device comprising:        -   i) at least one channel extending from a sample inlet to one            or more fluid outlets, wherein the channel is bounded by a            first wall and a second wall opposite from the first wall;        -   ii) an array of obstacles arranged in rows in the channel,            each subsequent row of obstacles being shifted laterally            with respect to a previous row, and wherein said obstacles            are disposed in a manner such that, when the crude fluid            composition is applied to an inlet of the device and            fluidically passed through the channel, T cells in the            composition flow to one or more collection outlets where an            enriched product is collected, and wherein platelets and            other materials smaller than T cells flow to one or more            waste outlets that are separate from the collection outlets;    -   c) genetically engineering the T cells in the enriched product        obtained in step b) to produce chimeric antigen receptors (CARs)        on their surface;    -   d) culturing the T cells to expand their number;    -   e) transferring the T cells into a pharmaceutical composition        for administration to a patient.-   E181. The method of E180, wherein at least 90% of platelets are    removed in step b).-   E182. The method of either E180 or E181, wherein all isolation steps    and concentration steps are carried out using DLD.-   E183. The method of any one of E180-182, wherein prior to, or during    culturing, cells are exposed to a T cell activator.-   E184. The method of any one of E180-183, wherein, in step b) and    prior to step c), cells are transferred into a medium in which a T    cell activator is present or added.-   E185. The method of E184, wherein the medium containing activator is    processed by DLD to separate activator from cells and to transfer    cells into a medium in which a vector for recombinantly engineering    cells is present or added.-   E186. The method of any one of E180-185, wherein cells are not    frozen until they are transferred into a pharmaceutical composition    for administration to a patient.-   E187. The method of any one of E180-185, wherein cells are not    frozen at any step.-   E188. The method of any one of E180-187, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least one day sooner.-   E189. The method of any one of E180-187, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least three days sooner.-   E190. The method of any one of E180-187, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least five days sooner.-   E191. The method of any one of E180-187, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least ten days sooner.-   E192. The method of any one of E180-191, wherein, in step d), the    number of T cells obtained after 14 days in culture is at least two    times higher than the number produced by the same procedure    performed using Ficoll centrifugation instead of DLD.-   E193. The method of any one of E180-191, wherein, in step d), the    number of T cells obtained after 14 days in culture is at least four    times higher than the number produced by the same procedure    performed using Ficoll centrifugation instead of DLD.-   E194. A method of preparing CART cells, comprising:    -   a) collecting cells from a patient by apheresis;    -   b) performing DLD on the cells obtained in step a) to separate        leukocytes from other cells and particles, and to transfer the        leukocytes into a medium that supports their growth and that        has, or is supplemented with, a T cell activator;    -   c) performing DLD to separate T cells from the medium of step b)        and to transfer cells into a medium where they are recombinantly        engineered to express chimeric antigen receptors (CARs) on their        surface;    -   d) performing DLD to separate the T cells from reagents and to        transfer the cells into a growth medium;    -   e) culturing the cells to expand their number    -   f) performing DLD to transfer expanded T cells into a medium for        administration to a patient.-   E195. The method of E194, wherein cells are not frozen until they    are transferred into a pharmaceutical composition for administration    to a patient.-   E196. The method of E194, wherein cells are not frozen at any step.-   E197. The method of any one of E194-196, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least three days sooner.-   E198. The method of any one of E194-196, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least five days sooner.-   E199. The method of any one of E194-196, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least ten days sooner.-   E200. A method of preparing CART cells for the treatment of a    patient comprising:    -   a) collecting cells from a patient by apheresis;    -   b) performing DLD on the cells obtained in step a) to separate        leukocytes from other cells and particles, and to transfer the        leukocytes into a medium that supports their growth and that        has, or is supplemented with, a T cell activator;    -   c) separating T cells from the medium of step b) into medium        that contains a vector for genetically engineering the T cells        to produce chimeric antigen receptors (CARs) on their surface;    -   d) performing DLD to separate T cells from reagents and to        transfer the cells into a medium for administration to a        patient.-   E201. The method of E200, wherein cells are not frozen until they    are transferred into a pharmaceutical composition for administration    to a patient.-   E202. The method of E200, wherein cells are not frozen at any step.-   E203. The method of any one of E200-202, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least three days sooner.-   E204. The method of any one of E200-202, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least five days sooner.-   E205. The method of any one of E200-202, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least ten days sooner.-   E206. The method of any one of E180-205, wherein in step b) a    product is obtained in which the ratio of platelets to leukocytes is    at least 50% lower than the ratio obtained using centrifugation or    elutriation instead of DLD.-   E207. The method of any one of E180-205, wherein the yield of T    cells exhibiting the desired CAR T phenotype is at least 10% greater    than identical cells isolated by Ficoll centrifugation and not    subjected to DLD.-   E208. The method of any one of E180-205, wherein the yield of T    cells exhibiting the desired CAR T phenotype is at least 20% greater    than identical cells isolated by Ficoll centrifugation and not    subjected to DLD.-   E209. The method of any one of E180-208, wherein the time necessary    to produce a sufficient number of T cells for the treatment of a    patient is at least 5% shorter than when the same method is carried    out using Ficoll centrifugation rather than DLD to isolate cells    from apheresis starting material.-   E210. The method of any one of E180-209, wherein the time necessary    to produce a sufficient number of T cells for the treatment of a    patient is at least 10% shorter than when the same method is carried    out using Ficoll centrifugation rather than DLD to isolate cells    from apheresis starting material.-   E211. The method of any one of E180-209, wherein, when cells    prepared by said method are administered to a patient, they exhibit    at least 10% less senescence than cells that have been processed    from an apheresis composition using centrifugation or elutriation    instead of DLD.-   E212. The method of any one of E180-209, wherein, when cells    prepared by said method are administered to a patient, they exhibit    increased efficacy when compared to cells that have been processed    from an apheresis composition using centrifugation or elutriation    instead of DLD.-   E213. A method for treating a patient for a disease or condition    comprising administering to said patient a therapeutically effective    amount of cells prepared by the method of any one of E180-209.-   E214. The method of E213, wherein said disease or condition is    cancer.-   E215. A method of producing therapeutically active cells,    comprising:    -   a) obtaining a sample composition from a patient comprising said        cells;    -   b) performing DLD on the sample to produce a composition        enriched in therapeutically active cells, wherein DLD is        performed on a microfluidic device comprising:        -   i) at least one channel extending from a sample inlet to one            or more fluid outlets, wherein the channel is bounded by a            first wall and a second wall opposite from the first wall;        -   ii) an array of obstacles arranged in rows in the channel,            each subsequent row of obstacles being shifted laterally            with respect to a previous row, and wherein said obstacles            are disposed in a manner such that, when the crude fluid            composition is applied to an inlet of the device and            fluidically passed through the channel, the therapeutically            active cells in the composition flow to one or more            collection outlets where an enriched product is collected,            and wherein cells and materials smaller than the            therapeutically active cells flow to one more waste outlets            that are separate from the collection outlets;    -   c) optionally genetically engineering the therapeutically active        cells in the enriched product obtained in step b);    -   d) optionally culturing the therapeutically active cells to        expand their number;    -   e) transferring the therapeutically active cells into a        pharmaceutical composition for administration to a patient.-   E216. The method of E215, wherein the therapeutically active cells    are stem cells.-   E217. The method of E216, wherein the stem cells are found in the    circulation and the sample composition is prepared by apheresis.-   E218. The method of E217, wherein at least 70% of platelets are    removed from the enriched product of step b).-   E219. The method of E217, wherein at least 90% of platelets are    removed from the enriched product of step b).-   E220. The method of any one of E215-219, wherein all isolation steps    and concentration steps are carried out using DLD.-   E221. The method of any one of E215-220, wherein cells are not    frozen until they are transferred into a pharmaceutical composition    for administration to a patient.-   E222. The method of any one of E215-220, wherein cells are not    frozen at any step.-   E223. The method of any one of E215-222, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least one day sooner.-   E224. The method of any one of E215-222, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least three days sooner.-   E225. The method of any one of E215-222, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least five days sooner.-   E226. The method of any one of E215-222, wherein the yield of    therapeutically active cells obtained from the sample composition is    at least 25% higher than the number obtained by a procedure in which    cells are isolated or concentrated by a method other than DLD.-   E227. The method of any one of E215-222, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least three days sooner.-   E228. The method of any one of E215-222, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    other than DLD, the process is completed at least five days sooner.-   E229. The method of any one of E215-222, wherein the time necessary    to produce a sufficient number of therapeutically active cells for    the treatment of a patient is at least 5% shorter than when the same    method is carried out using Ficoll centrifugation rather than DLD to    isolate cells from the sample composition.-   E230. The method of any one of E215-222, wherein the time necessary    to produce a sufficient number of therapeutically active cells for    the treatment of a patient is at least 10% shorter than when the    same method is carried out using Ficoll centrifugation rather than    DLD to isolate cells from the sample composition.-   E231. The method of any one of E215-230, wherein, when the    therapeutically active cells prepared by said method are    administered to a patient, they exhibit at least 10% less senescence    than cells that have been processed from an apheresis composition    using centrifugation or elutriation instead of DLD.-   E232. The method of any one of E215-230, wherein, when the    therapeutically active cells prepared by said method are    administered to a patient, they exhibit increased efficacy when    compared to cells that have been processed from the sample    composition using centrifugation or elutriation instead of DLD.-   E233. A method for treating a patient for a disease or condition,    comprising administering to said patient a therapeutically effective    amount of the therapeutically active cells prepared by the method of    any one of E215-230.-   E234. The method of E233, wherein said the therapeutically active    cells are stem cells and the disease or condition is a genetic    disease.-   E235. A method of engineering a population of target cells,    comprising:    -   a) isolating the target cells from a crude fluid composition        wherein isolation is carried out on a microfluidic device, using        one or more procedures which separate target cells from other        cells in the crude fluid composition based on differences in        size and wherein the isolation produces a composition enriched        in target cells;    -   b) genetically engineering the target cells obtained from        step a) to have a desired phenotype;    -   wherein target cells are not centrifuged or elutriated prior to        being genetically engineered.-   E236. The method of E235, wherein said target cells are leukocytes    or stem cells.-   E237. The method of E235, wherein said target cells are T cells.-   E238. The method of any one of E235-237, wherein said crude fluid    composition is blood or an apheresis preparation obtained from a    patient.-   E239. The method of E238, wherein isolation of target cells takes    place under conditions such that the composition enriched in target    cells has a total number of platelets that is at least 70% lower    than in the apheresis preparation.-   E240. The method of E239, wherein the total number of platelets in    the composition enriched in target cells is at least 90% lower than    in the apheresis preparation.-   E241. The method of E240, wherein the ratio of platelets to target    cells is at least 50% lower than in the apheresis preparation.-   E242. The method of E238, wherein said target cells are T cells    that, after isolation are expanded in cell culture.-   E243. The method of E242, wherein the number of T cells obtained    after 14 days in culture is at least two times higher than in a    procedure in which T cells are isolated by a process including a    centrifugation step.-   E244. The method of E242, wherein the number of T cells obtained    after 14 days in culture is at least four times higher than in a    procedure in which T cells are isolated by a process including a    centrifugation step.-   E245. The method of E244, wherein the percentage of memory T cells    in culture relative to the total number of T cells is at least 10%    higher than in a procedure in which T cells are isolated by a    process including a centrifugation step.-   E246. The method of E244, wherein the percentage of memory T cells    in culture relative to the total number of T cells is at least 20%    higher than in a procedure in which T cells are isolated by a    process including a centrifugation step.-   E247. The method of any one of E235-246, wherein, when cells in the    composition enriched in target cells are transformed with a vector    to express a recombinant phenotype, the yield of target cells    exhibiting the desired phenotype is at least 20% greater than for    identical cells isolated by centrifugation.-   E248. The method of any one of E238-247, wherein no more than one    hour elapses from the time that apheresis sample collection is    completed until the time that separation using the microfluidic    device is performed.-   E249. The method of any one of E238-247, wherein no more than four    hours elapse from the time the obtaining of the apheresis sample    from the patient is completed until the isolation of target cells is    completed.-   E250. The method of any one of E238-247, wherein no more than four    hours elapse from the time the obtaining of the apheresis sample    from the patient is completed until cells are genetically    engineered.-   E251. A method of producing CART cells, comprising:    -   a) obtaining a crude fluid composition from a patient by        apheresis, wherein said sample composition comprises T cells;    -   b) isolating the T cells from the crude fluid composition,        wherein isolation is carried out on a microfluidic device using        one or more procedures which separate T cells from platelets and        other cells in the crude fluid composition based on differences        in size and wherein the isolation produces a composition        enriched in T cells and depleted in platelets;    -   c) genetically engineering the T cells obtained from step a) to        produce chimeric antigen receptors (CARs) on their surface, and        wherein the T cells are not centrifuged or elutriated at any        step prior to being genetically engineered;    -   d) culturing the genetically engineered T cells to expand their        number;    -   e) collecting the cultured cells produced in step d).-   E252. The method of E251, wherein, in step e), T cells are collected    by being transferred into a pharmaceutical composition for    administration to a patient.-   E253. The method of E251 or 252, wherein cells are not frozen before    being collected.-   E254. The method of any one of E251-253, wherein at least 90% of    platelets are removed in step b).-   E255. The method of any one of E251-254, wherein prior to, or during    culturing, cells are exposed to a T cell activator or a carrier.-   E256. The method of E255, wherein neither said activator nor said    carrier are bound to a magnetic bead or particle.-   E257. The method of any one of E251-256, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    not involving the use of a microfluidic device, the CAR T cells are    available for administration to a patient at least one day earlier.-   E258. The method of any one of E251-256, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    not involving the use of a microfluidic device, the CAR T cells are    available for administration to a patient at least three days    earlier.-   E259. The method of any one of E251-258, wherein, in step d), the    number of CAR T cells obtained after 14 days in culture is at least    two times higher than the number produced by cells obtained using    Ficoll centrifugation.-   E260. The method of any one of E251-258, wherein, in step d), the    number of CAR T cells obtained after 14 days in culture is at least    four times higher than the number produced by the same procedure    performed using Ficoll centrifugation.-   E261. The method of any one of E251-260, wherein, T cells and CAR T    cells are never frozen before being administered to a patient.-   E262. A method of preparing CAR T cells for the treatment of a    patient comprising:    -   a) obtaining a crude fluid composition from the patient by        apheresis, wherein said composition comprises T cells;    -   b) isolating the T cells from the crude fluid composition,        wherein isolation is carried out on a microfluidic device using        one or more procedures which separate T cells from platelets and        other cells in the crude fluid composition based on differences        in size and wherein the isolation produces a composition        enriched in T cells and depleted in platelets;    -   c) genetically engineering the T cells obtained from step a) to        produce chimeric antigen receptors (CARs) on their surface, and        wherein the T cells are not centrifuged or elutriated at any        step prior to being genetically engineered;    -   d) culturing the genetically engineered T cells to expand their        number;    -   e) separating T cells on a microfluidic device using one or more        procedures which separate T cells from reagents based on        differences in size.-   E263. The method of E262, wherein cells are not frozen until they    are transferred into a pharmaceutical composition for administration    to a patient.-   E264. The method of E262, wherein cells are not frozen at any step.-   E265. The method of any one of E262-264, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    that does not use a microfluidic device, the process is completed at    least three days sooner.-   E266. The method of any one of E262-265, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    not involving the use of a microfluidic device, the CAR T cells are    available for administration to a patient at least one day earlier.-   E267. The method of any one of E262-265, wherein, compared to a    procedure in which cells are isolated or concentrated by a method    not involving the use of a microfluidic device, the CAR T cells are    available for administration to a patient at least three days    earlier.-   E268. The method of any one of E262-267, wherein in step b) a    product composition is obtained in which the ratio of platelets to    leukocytes is at least 50% lower than the ratio obtained using    centrifugation or elutriation.-   E269. The method of any one of E262-268, wherein the time necessary    to produce a sufficient number of CAR T cells for the treatment of a    patient is at least 5% shorter than when the same method is carried    out using cells isolated by Ficoll centrifugation.-   E270. The method of any one of E262-269, wherein the time necessary    to produce a sufficient number of CAR T cells for the treatment of a    patient is at least 10% shorter than when the same method is carried    out using Ficoll centrifugation to isolate cells from apheresis    starting material.-   E271. The method of any one of E262-270, wherein, when cells    prepared by said method are administered to a patient, they exhibit    at least 10% less senescence than cells that have been processed    from an apheresis composition using centrifugation or elutriation.-   E272. A method for treating a patient for a disease or condition    comprising administering to said patient a therapeutically effective    amount of cells prepared by the method of any one of E235-271.-   E273. The method of E272, wherein said disease or condition is    cancer.

EXAMPLES

The following examples are intended to illustrate, but not limit theinvention.

Example 1

This study focuses on apheresis samples, which are integral toCAR-T-cell manufacture. The inherent variability associated with donorhealth, disease status and prior chemotherapy all impact the quality ofthe leukapheresis collection, and likely the efficacy of various stepsin the manufacturing protocols (Levine, et al., Mol. Therapy: Meth.Clin. Dev. 4:92-101 (2017)). To stress test the automated DLD leukocyteenrichment, residual leukocytes (LRS chamber fractions) were collectedfrom plateletpheresis donations which generally have near normalerythrocyte counts, 10-20-fold higher lymphocytes and monocytes andalmost no granulocytes. They also have ˜10-fold higher platelet counts,as compared to normal peripheral blood.

12 donors were processed and yields were compared of major blood celltypes and processivity by DLD versus Ficoll-Hypaque density gradientcentrifugation, a “gold standard.” 4 of these donors were also assessedfor “T-cell expansion capacity” over a 15-day period. Each donor samplewas processed by both DLD, and Ficoll, and for the 4 donors studied forT-cell expansion capacity the sample was processed using direct magneticextraction.

Materials and Methods

Microchip Design and Fabrication:

The DLD array used in this study consisted of a single-zone, mirrored,diamond post design (see D'Silva, J., “Throughout Microfluidic Captureof Rare Cells from Large Volumes of Blood;” A Dissertation Presented tothe Faculty of Princeton University in Candidacy for the Degree ofDoctor of Philosophy (2016)). There were 14 parallel arrays per chipresulting in a 14-lane DLD device (FIG. 1D). The device was designedwith a 16 μm gap between posts and a 1/42 tilt, resulting in a criticaldiameter of ˜4 μm. The plastic DLD device was generated using a processcalled soft-embossing. First, a silicon (Si) master for the plastic DLDmicrochip was made using standard photolithographic and deep reactiveion etching techniques (Princeton University, PRISM). The features onthe silicon master were then transferred to a soft elastomeric mold(Edge Embossing, Medford, Mass.) by casting and curing the elastomerover the Si features. The elastomer was peeled off to create a reusable,negative imprint of the silicon master. A plastic blank sheet was placedbetween the elastomer molds, and then using a combination of pressureand temperature, the plastic was extruded into the features (wells) ofthe soft-elastomer negative mold, replicating the positive features anddepth of the original silicon master. The soft tool was then peeled offfrom the plastic device, producing a flat piece of plasticsurface-embossed to a depth ˜100 μm with a pattern of flow channels andtrenches around an array of microposts (FIG. 1D, inset). Ports werecreated for fluidic access to the Input and Output ends of themicrochip. After cleaning by sonication, the device was lidded with aheat-sensitive, hydrophilic adhesive (ARFlow Adhesives Research, GlenRock, Pa.). The overall chip was 40×75 mm, and 1 mm thick—smaller thanthe size of a credit card.

DLD Microchip Operation:

The microfluidic device was assembled inside an optically transparentand pressure resistant manifold with fluidic connections. Fluids weredriven through the DLD microchip using a constant pneumatic pressurecontroller (MFCS-EZ, Fluigent, Lowell, Mass.). Two separate pressurecontrols were used, one for buffer and one for sample. The flow path forthe buffer line included tubing connecting a buffer reservoir (60 mLsyringe), an in-line degasser (Biotech DEGASi, Minneapolis, Minn.) andthe buffer inlet port of the manifold. The flow path for the sampleincluded tubing connecting a sample reservoir (20 mL syringe), a 20 μmPureFlow nylon filter of 25 mm diameter (Clear Solutions, Inc. SanClemente, Calif.) to retain aggregates larger than the microchipsnominal gap size (16 μm), and the sample inlet port on the manifold. Theoutlet ports of the manifold were connected by tubing to collectionreservoirs for the waste and product fractions.

The microchips, filter and tubing were primed and blocked for 15 minwith running buffer before the sample was loaded. The DLD setup wasprimed by loading running buffer into the buffer reservoir (60 mLsyringe) and then pressurizing; fluid then passed through the tubing andinto the manifold “Buffer in” port (FIG. 1). Air in the manifold portwas vented via another port on that inlet, and then that port wassealed. The buffer was then driven through the microchip and out boththe product and waste outlets, evacuating all air in the micropostarray. At the same time, buffer was back flushed up through the “SampleIN” port on the manifold and through the in-line filter, flushing anyair. This priming step took ˜5 min of hands-on time, and removed all airfrom the microchip, manifold and tubing. Following the prime step,buffer continued to flush the setup for an additional 15 minutes toblock all the interior surfaces; this step was automated and did notrequire hands-on time.

Following the block step, the system was depressurized, and sample wasloaded into the sample container (20 mL syringe). The sample (see below)was diluted 1-part sample to 4 parts running buffer (0.2×) prior toloading on the DLD. The buffer source was re-pressurized first, then thesample source, resulting in both buffer and sample entering theirrespective ports on the manifold and microchip and flowing through themicrochip in parallel (see separation mode, FIG. 1 Ai). Once the samplewas loaded and at running pressure, the system automatically processedthe entire sample volume. Both product and waste fractions werecollected in pre-weighed sterile conical 50 mL tubes and weighed afterthe collection to determine the volumes collected.

Buffer Systems.

Three different EDTA free buffer formulations were tested on the DLD:0.5% F127 (Pluronic F-127, Sigma Aldrich, St. Louis, Mo.) inphosphate-buffered saline [Ca⁺⁺/Mg⁺⁺ free) (Quality biological,Gaithersburg, Md.), 1% Bovine Serum Albumin (BSA) (Affymetrix, SantaClara, Calif.) in phosphate-buffered saline [Ca⁺⁺/Mg⁺⁺ free], and anisotonic Elutriation Buffer (EB) composed of 50% Plasmalyte A (Baxter,Deerfield, Ill.) and 50% of a mixture containing 1.0% BSA (Affymetrix,Santa Clara, Calif.) 1.0 mM N-Acetyl-Cysteine, 2% Dextrose and 0.45%NaCl (all from Sigma-Aldrich, St. Louis, Mo.). The buffers were preparedfresh each day, and were sterile-filtered through a 0.2 μm filter flaskprior to use on the DLD. All samples in the expansion group wereprocessed using the isotonic elutriation buffer to best align withcurrent CAR-T-cell manufacturing approaches, even though better DLDperformance has been established with the addition of poloxamer(Johnson, et al., Cancer Cell Res. 27:38-58 (2017)).

Biological Samples.

Leucoreduction System (LRS) chamber samples from plateletpheresisdonations of normal screened donors using a Trima system (Terumo, Tokyo,Japan) were obtained from the local blood bank. Cell counts were done atthe time of collection by the blood bank. Counts were verified in ourlab, using a Beckman Coulter AcT2 Diff2 clinical blood analyzer, andranged between 76-313.3×10³ WBC/μL and 0.8-4.87×10⁶ platelets/μL. Allsamples were kept overnight at room temperature on an orbital shaker(Biocotek, China), and then processed the following day (˜24 hourslater) to mimic overnight shipment. Each donor sample was processed byboth DLD, and Ficoll, and for the 4 donors used for T-cell expansion andimmunophenotypic studies the sample was also processed using directmagnetic extraction.

Ficoll-Hypaque.

Peripheral blood mononuclear cells (PBMCs) were obtained by diluting theLRS sample to 0.5× in RPMI (Sigma-Aldrich. St Louis, Mo.), layered ontop of an equal volume of Ficoll-Hypaque (GE, Pittsburgh, Pa.) in a 50mL conical tube, and centrifuged for 35 min with a free-swinging rotor,and no brake, at 400×g. After centrifugation, the top layer wasdiscarded and the interface PBMC fraction transferred to a new 50 mLtube and brought up to 20 mL of RPMI. PBMCs were washed bycentrifugation for 10 min at 400×g, the supernatant discarded and thepellet resuspended with 20 mL of RPMI and washed again at 200×g for 10min. The supernatant was removed and the pellet resuspended in fullmedia containing RPMI-1640+10% Fetal Bovine Serum (FBS) (Sigma-Aldrich,St. Louis, Mo.) plus penicillin 100 units/mL and streptomycin 100 μg/mLantibiotics (Thermo-Fisher, Waltham, Mass.).

Cell Isolation, Counting, and Immunofluorescence Staining.

Prior to and after isolation using the methods described above, the cellcounts of the resulting products were determined using a blood cellanalyzer (Beckman-Coulter AcT2 Diff2). Once in culture, and afteractivation, cell counts were determined using the Scepter™ 2.0 hand-heldcell counter (Millipore, Billerica, Mass.) and by absolute countingusing flow cytometry. Cells from the input, product and waste fractionswere then loaded onto poly-lysine-coated slides for 10 min and thenfixed for 15 min in 4% p-formaldehyde+0.5% Triton X-100 in PBS, beforewashing 3 times in PBS by centrifugation. Slides were incubated with theconjugated primary antibodies CD41-A647 and CD41-FITC (both fromBioLegend San Diego, Calif.) for 60 min in the dark and washed threetimes with PBS before mounting in slow-fade mounting media containingthe DNA stain DAPI (Thermo-Fisher, Waltham, Mass.). Slides were viewedwith an Etaluma™ Lumascope 620 fluorescence inverted microscope(Carlsbad, Calif.). Antibodies (mAb) conjugated to fluorochromes wereobtained from BioLegend (San Diego, Calif.): CD25-PE, CD25-APC,CD95-FITC, CD45RA-BV605, CD45RO-PECy7, CD197/CCR7 PE, CD279-PE, CD28PE-Cy5, CD45-PerCP, CD3-FITC, CD3-BV421, CD4-AF700, CD8-APC-AF780,CD61-FITC, CD41-FITC, CD45-Alexa647. Viability of the WBCs obtained byDLD and PBMCs purified by Ficoll-Hypaque was determined by Trypan blueexclusion.

Activation and Magnetic Separation.

For T-cell stimulations in expansion group, DLD, Ficoll and LRS productwere diluted to 1×10⁷ T cells/mL then activated with washed andequilibrated anti-CD3/CD28 conjugated magnetic beads (5.0 μm)(Thermo-Fisher, Waltham, Mass.) at a ratio of 3.2:1 beads per cell for60 min, and then the activated T cells were separated by a magneticdepletion for 5 min. Unbound cells were removed, and the bead-boundcells were cultured further in full media (below). In the direct magnetprotocol, 0.5 mL of LRS sample (same donor as was processed via DLD orFicoll) was incubated with immunomagnetic CD3/CD28 beads for one hour.The mixture was then placed against a magnet for 5 minutes to capturethe T cells. The magnetic bead-bound cells (activated cells) wereremoved and then diluted to 0.5×10⁶/mL as above for culture in fullmedia.

After three days in culture, recombinant human IL-2 (BioLegend, SanDiego, Calif.) was added at 200 IU/mL to wells. Following cell culturefor up to 15 days, beads were removed from cells and cells counted ateach time point. To remove beads, the cells in the well were resuspendedby passing the cells through a 5-mL pipette for 10 times. Next, the cellsuspension was passed throughout a 1 mL pipette 40 times followed byvigorous pipetting using a 200 μL tip for 1 min. Then the cellsuspension was placed on the side of a magnet for 5 min and thenonmagnetic fraction was transferred to a fresh tube and counted. Thenumber of cells in the culture wells was determined using a Scepterhand-held cell counter and by flow cytometry.

Cell Culture and Cell Activation.

For each of the T-cell preparations put into cell culture, in additionto the stimulated cells described above, unstimulated cells (controls)were adjusted to 0.5×10⁶/mL in complete media (RPMI+10% FBS+antibiotics)and plated in 6-well plates (Corning, N.Y.) and cultured at 37° C., 5%CO₂ in a humidified incubator. Individual wells, for each condition,unstimulated, and stimulated with and stimulated without IL2, werededicated to each donor at each time point to eliminate any possibilityof disruption in expansion due to sampling and the de-beading activityrequired for reliable counts, particularly at Day 3.

Flow Cytometry.

No-wash absolute counting by flow cytometry was used for CD3+ cellcounts at all time points, Initial day 0 counts used TruCount tubes (BDBiosciences, San Jose, Calif.) to accurately determine the number ofcells recovered and counted. Subsequent days used 25,000 123 beads(Affymetrix, Santa Clara, Calif.) which were indexed against TruCounttubes as an internal control. 100 μL of a cell suspension was stainedwith the CD3 FITC, CD25 PE and CD45 PerCP of conjugated antibodies for30 min in the dark in either TruCount tubes or with addition of 25,000123 beads (Affymetrix, Santa Clara, Calif.). The cells were then dilutedto 250 μL of PBS with a final DRAQ5™ DNA dye (Thermo-Fisher, Waltham,Mass.) concentration of 1.0 mM. Next, the stained cells were fixed withan additional 250 μL 1.2% p-formaldehyde in PBS overnight prior toacquisition. For absolute count cytometry, a minimum of 25,000 events or2500 bead events were acquired on a BD FACSCalibur (BD Biosciences, SanJose, Calif.) using a fluorescence threshold (CD45 PerCP). Phenotypicanalysis was also performed at all time points, using a 7-coloractivation/anergy panel consisting of CD3, CD45RA, CD95, CD279, CD25,CD4, and CD8. At day 15 the panel was modified to create a 9-color panelfocused on T central memory cells which added CD45RO PE-Cy7, CD28 PE-Cy5and substituted CD197/CCR7 PE for CD279/PD1 PE. For multicolor staining,100 μl of a cell suspension was stained as above, and resuspended in 750μL PBS and washed by centrifugation at 400×g and then resuspending in250 μL 1.2% p-formaldehyde and fixed overnight prior to acquiring 20,000events using forward scatter threshold on a four laser BD FACSAria II.(BD Biosciences, San Jose, Calif.). All data analysis was performedusing Flowlogic Software (Inivai, Melbourne, Australia).

Results

DLD Microchip and Ficoll Processing of Apheresis Products

The DLD and Ficoll separation methods were used to process 12 LRSsamples obtained from 12 separate normal donors. Of those 12 samplesreceived and processed, 11 samples clustered around a mean of148.7×10³/μL WBC and 2.52×10⁶/μL platelet counts respectively (FIG. 2A,2B). The 12^(th) sample, with 313.3×10³/μL WBC and 4.87×10⁶/μL plateletcounts can be seen in the scatter plot as a red triangle, (FIG. 2A).This sample was sufficiently aggregated at the time of processing thatit rapidly clogged the 20 μm prefilter and thus did not fully enter theDLD. Microscopic examination of the input sample showed that this samplewas full of platelet-WBC aggregates ranging in size from 25-50 μm withmultiple aggregates observed as large as 250 μm in diameter (FIG. 2C,2D). Further, both WBC and platelet counts were greater than 3 standarddeviations above the mean WBC and platelet count. Using the quartilemethod, this sample was classified as a mild outlier; using the Grubbstest for outliers and an alpha level of 0.05, this sample was alsoclassified as an outlier.²⁰ As a result, this donor was excluded fromthe study based on extremely high WBC and platelet counts and being toobadly agglutinated and damaged.

A representative image of the input material (LRS product diluted to0.2×) is shown in (FIG. 2A). Typical micrographs of DLD (FIG. 2E) andFicoll (FIG. 2C) cell products from the same input donor, withsignificantly lower background platelet levels (CD41-FITC in green)found in the DLD compared to Ficoll. Also shown are the respective cellproducts, as collected in tubes (FIG. 2 G, H). DLD processing automatedthe process of removing the WBCs from the RBCs and platelets, generatingone tube for product and one for waste, while the Ficoll sample stillrequires further manual processing to pipet the PMBC layer at theoperationally-defined interface of the plasma layer above and Ficolllayer below (FIG. 2H); plus, an additional minimum of two centrifugalwashes are required to remove most of the contaminating platelets.

The recovery of WBC, and RBC and platelet depletions of the 11 samplesare summarized in Table 2. Mean cell recoveries of PBMC from DLD were˜80%. 17% higher than Ficoll (63%), and, after accounting for the numberof CD3 cells in both the DLD and magnetic samples, the DLD product was36% higher than Direct Magnet (44%). Mean platelet depletion via DLD(83%) was superior to both Ficoll (56.5%) and direct magnet (77%). Meanerythrocyte depletion in these 24-hour old samples was 97% for both DLDand Ficoll, and 94% for the direct magnet approach. The averageviability of cells obtained by DLD was 96% compared to Ficoll which were97%.

The average total time taken to process equivalent aliquots of a singlesample in a 50 mL conical tube via the Ficoll technique was timed at ˜90minutes, with approximately 30 minutes of skilled hands-on timerequired. Timed runs using our single microchip layer breadboard systemprocessed in much shorter time, 50 minutes and required 25 minutes ofhands on time, with approximately 20 minutes being due solely toassembly of fluidics components because of the prototypic nature of theotherwise intervention free device.

Cell Expansion and Characterization

Following DLD or Ficoll enrichment, cells were activated using CD3/CD28magnetic beads for 60 minutes at a target of 3.2 beads per CD3+ cell,separated and then counted prior to plating. Due to limited access to aflow cytometer, and concerns regarding potential bead interference inproduct cell counts, we estimated the T cell count by counting both theinput and non-magnetic fraction and getting the number of T cells boundto the magnet by subtraction, using an assumption of a 90% efficientmagnetic separation (based on manufacturer reported efficiencies).Accurate T-cell counts were determined post-plating into culture usingabsolute counts by flow cytometry and by coulter counts x % CD3 positivecells; these counts established that the original magnetic CD3+ celldepletion process was only 44% efficient (Table 2). This meant thatoriginal calculations pertaining to a target of 3.2 beads per CD3+ cellwere in fact on average 2.3 for both the DLD and Ficoll fractions (fewerbeads per T-cell than targeted), and a 5:1 ratio in the direct magnetfraction (significantly more beads per T-cell than targeted),potentially causing the direct magnet fraction to have even higher foldexpansion compared to both the DLD and Ficoll arms.

Flow cytometric characterization of the cultures was performed at eachtime point to assess consistency of cell activation. Changes in CD25expression of CD3+ cells, as measured on Day 8, for Ficoll, DLD anddirect magnet (FIG. 3). IL-2 Receptor positive (CD25) CD3 cells areshown in Blue (CD4+ plots) and Red (CD8+ plots). DLD prepared cells showmore consistent phenotypic expression across the 4 donors for CD25, anindicator of response to CD3/CD28 stimulation, as compared to bothFicoll and direct magnet preparations. DLD prepared CD3+ cells had anaverage 73% response to co-stimulation compared to Ficoll at 51% (bothstimulated at 2.3 beads/cell), while the direct magnet fraction,stimulated at a higher 5:1 ratio, had only a 54% response.

Unstimulated controls for Ficoll and DLD show a marked difference, withDLD prepared cells remaining CD25 negative in culture compared to Ficoll(FIG. 9). Interestingly, Donor 37 in the direct magnet fraction did notrespond by day 8, but did expand at later time points (also shown in(FIG. 5A)) indicating a potentially delayed response of some samples tothe direct magnetic approach.

In addition to evaluating CD25, conversion to a memory cell phenotypewas tracked using percentage of CD3+ cells that were CD45RA- and CD25+.The results shown in FIG. 4 indicate a greater percentage of thecultured cells, as generated via DLD, were responsive to co-stimulationcompared to cells processed by Ficoll and direct magnetics. Further, thepercent of CD3 cells that were CD25− CD45RA− was lowest in the DLDfraction at 12% as compared to 33 and 29% for Ficoll and Direct Magnetrespectively, indicating a more complete conversion towards theCD25+CD45RA− population with the DLD CD3 cells. The standard deviationof the CD45RA−CD25+ population at day 8 for DLD was 10.1% as compared to24.8% for Ficoll and 53.4% for Direct Magnet.

The fold expansion of the individual cultures was determined at day 3,day 8 and day 15; that data is shown in FIG. 5A. The plot shows theexpansion of each donor sample, across each method. While the directmagnet approach appears to show higher expansion, the counts are likelysignificantly affected by the different bead:cell ratios (andcorresponding differences in plating density). Regardless, the 4 donorsshow significant variability in the fold expansion. In addition, the day15 culture for the direct magnet arm donor #21 became contaminated andhad to be discarded, despite having antibiotics present. It is notpossible to know if the day 8 expansion data for donor #21 wereinfluenced by the contaminant.

Comparisons between the Ficoll and DLD are valid and much more direct:these cells were plated at the same density and stimulated at the samebead:cell ratio. While the average fold expansion of the DLD cells isnot significantly higher than that of the Ficoll cells, the consistencyof expansion across the set of 4 donors, and at all days surveyed, isstriking. Further the percent of cells in culture that are a centralmemory phenotype is on average 74% for the DLD arm, contrasted to 47%and 48% respectively for the Ficoll and Direct Magnet arms. Multiplyingfold expansion in 5A by percent yield (table 1) and percent memory (FIG.5B) shows that, despite the sub optimal comparison with bead:cellratios, that on average twice as many memory cells were produced fromthe DLD arm as compared to either Ficoll or Direct Magnet arms.

FIG. 6 shows the phenotypic approach to identifying memory cells used inthis study, which is designed to eliminate any issues with shed antigenssuch as CD62L (Mahnke, et al., Eur. J. of Immunol. 43:2797-2809 (2013)).Central memory cells are sequentially gated and then backgated to showthe CD3+ T cells are positive for CD45R0+, CD95+, CD28+ and CD197/CCR7+against all other CD3+ cells in the culture. Using an arbitrary greaterthan 50% of the culture as being a central memory phenotype as aconversion metric, the DLD arm showed 100% (4/4) donors achievingcentral memory conversion with an average of 74% of cells being ofmemory phenotype, with coefficient of variation across donors of 13%. Incontrast, the Ficoll arm showed 50% (2/4) converting with an average of47% memory cells, and a 29% variation. The direct magnet arm achieved33% (1/3) conversion with an average of 48% memory cells and anassociated 79% variation.

TABLE 2 Comparison of DLD, Ficoll and Direct Magnetic Enrichment WBC RBCPlatelet Recovery Depletion Depletion DLD (n = 11) Average 79.6% 96.9%83.1% STDEV 13.4%  1.1% 12.3% Range 46.5-93.7% 95.5-98.6%  60.5-100.0%Median 80.1% 97.0% 87.6% Ficoll (n = 11) Average 63.5% 97.1% 56.5% STDEV16.3%  1.7% 22.8% Range 22.4-83.7% 94.1-99.9% 67.0-92.1% Median 65.6%97.0% 52.3% Direct Magnet (CD3 positive) (n = 4) Average 44.0% 94.1%77.6% STDEV  5.8%  3.3% 10.4% Range 36.8-50.7% 90.1-97.6% 25.0-99.1%Median 65.6% 94.5% 76.0%

Example 2: Platelet Add Back Experiment

Rationale

Previously, it has been found that WBC derived from the DLD isolationand purification are healthy and responsive to activation by CD3/CD28antibodies and differentiate towards their Tcm (T central memory)phenotype (Campos-Gonzalez, et al., SLAS, Jan. 23, 2018, publishedonline doi.org/10.1177/2472630317751214). Additionally, in the presenceof IL-2 Tcm cells expand and proliferate accordingly and similarly tocells derived from other methods, like Ficoll.

A key feature of the DLD cell purification is the efficient removal ofred blood cells and platelets to provide a highly purified white bloodcells (WBC) product. In comparison, Ficoll-derived white blood cells(PBMC's) show more contaminating red blood cells and platelets dependingon the sample quality. On average the platelet “contamination” in theFicoll-derived cells is 44% has a range of about 22% of variabilitywhereas the DLD cells exhibit only a 17% platelet contamination withvariability of +/−12%.

Because of the striking differences in the platelet depletion inDLD-processed Apheresis blood when compared to the Ficoll-separatedApheresis blood, an investigation was made of whether the addition ofautologous platelets to the DLD-purified white blood cells affects theproliferation and Tcm production over a period of time.

Experimental Details

Two different Leukoreduction System-apheresis (“LRS-apheresis”) sampleswere collected from a local blood bank either as controls or in 2.0 mMEDTA. All four samples were processed identically by two differentmethods in parallel: DLD processing and Ficoll gradient centrifugation.The original platelet:WBC ratios provided by the blood bank wereannotated and confirmed by coulter counter determinations.

3.0 ml of each LRS blood were processed by DLD according to ourpreviously described protocol by diluting the blood to 0.2× with 1.0%BSA/5.0 mM EDTA in PBS. Samples were run with a 1.0% BSA/PBS bufferunder standard pressure and condition using an individual DLD-14 lanechip for each sample. Product and waste were collected, and thecellularity was measured using a coulter counter.

3.0 ml of each LRS blood product from the two different donors andconditions (collected in 2.0 mM EDTA or control) were diluted 1:1 with3.0 ml of Phosphate-buffered saline (minus Calcium and Magnesium) andlayered on top of 6.0 ml of Ficoll-Paque in a 50 ml conical tube. Theperipheral mononuclear cells of PBMC's were obtained by centrifugationfor 35 min at 400×g with no brake. The top layer, or plasma-richfraction, was removed and transferred to another tube and diluted 1:1with PBS/1.0%/BSA. The PBMC were washed with an excess of PBS bycentrifugation, once at 400×g for 10 min and the second time at 200×gfor 10 min. Both supernatants were transferred to new 50 ml conicaltubes and diluted 1:1 with PBS/1.0% BSA. The diluted plasma-richfraction and the two supernatants were centrifuged at 1,200×g for 15min, the supernatants discarded, and the pellets resuspended in PBS/1.0%BSA, combined and centrifuged once more at 1,200×g for 15 min.Supernatant was discarded and the pellet-or plateletfraction-resuspended in 1.0 ml of PBS/1.0% BSA by gentle pipetting. Theplatelets were measured using the coulter counter. The correspondingplatelets were added back to the DLD-derived WBC at the desired ratiosand incubated for 1 h before activation with CD3/CD28 magnetic beads(Thermo Fisher).

After activation, the cells were placed in complete RPMI media+10%FBS+antibiotics and cultured over set times in a humidified incubator at37° C. and 5% CO₂. Cell aliquots were analyzed at days 3, 7, and 14 bymulti-color flow cytometry using the combination of antibodies indicatedin the figures. Cell culture aliquots were obtained at the differentdays and the cells were de-beaded as previously described beforepreparation for flow cytometry. Also, cell proliferation was measured byusing a Scepter manual cell counter. We then compared the differencesbetween the Ficoll-derived cells with those obtained from the DLDprocessing under control conditions and when platelets—at differentratios—were added back to the DLD-cells. The parameters we used were thenumber of cells at the different time points and the number of Tcm cellsaccording to their phenotype by flow cytometry.

The scheme below illustrates the overall experimental design followedduring this experiment with steps proceeding from top to bottom.

SAMPLE SEPARATION

PLT ADD BACK + + + + CD3/CD28 + + + + + + + + (ACTIVATION)

Results and Conclusions

White blood cells obtained using DLD consistently showed less plateletsthan white blood cells obtained using Ficoll (see FIGS. 19-21). Theresults also demonstrate a clearly superior expansion of T cells derivedfrom the DLD as compared to their counterparts from Ficoll (FIG. 22).Furthermore, the addition of platelets back to the DLD-isolated cellsreduced their ability to expand to the same levels as the platelet-freeDLD cells (FIG. 22). These results support the hypothesis that the moreefficient platelet-reduction during DLD processing of blood productsproduces white blood cells more responsive to activation by CD3/CD28 andexpansion by IL-2.

REFERENCES

-   1. Vonderheide, R. H., and June, C. H. Engineering T-cells for    Cancer: Our Synthetic Future. Immunol. Rev. 2014, 257, 7-13.-   2. Fousek, K., and Ahmed, N. The Evolution of T-cell Therapies for    Solid Malignancies. Clin. Cancer Res. 2015, 21, 3384-3392.-   3. Wang, X and Riviere, I. Clinical Manufacturing of CAR-T-cells:    Foundation of a Promising Therapy. Mol. Ther. Oncolytics 2016, 3,    16015.-   4. Sadelain, M., Riviere, I. and Riddell, S. Therapeutic T-cell    engineering. Nature, 2017, 545, 423-431.-   5. National Cell Manufacturing Consortium. Achieving Large-Scale,    Cost-Effective, Reproducible Manufacturing of High Quality Cells. A    Technology Roadmap to 2025. February 2016.-   6. Levine, B. L., Miskin, J., Wonnacott, K., et al. Global    Manufacturing of CAR T-cell Therapy. Mol. Therapy: Meth. Clin. Dev.    2017, 4, 92-101.-   7. Couzin-Frankel, J. Supply of Promising T-cell Therapy is    Strained. Science 2017, 356, 1112.-   8. Johnson, L. A., and June, C. H. Driving Gene-engineered T-cell    Immunotherapy of Cancer. Cell Res. 2017, 27, 38-58.-   9. Hokland, P., and Heron, I. The Isopaque-Ficoll Method    Re-evaluated: Selective Loss of Autologous Rosette-forming    Lymphocytes During Isolation of Mononuclear Cells from Human    Peripheral Blood. Scand. J. Immunol. 1980, 11, 353-356.-   10. Stroncek, D. F., Fellowes, V., Pham, C., et al. Counter-flow    Elutriation of Clinical Peripheral Blood Mononuclear Cell    Concentrates for the Production of Dendritic and T-cell    Therapies. J. Transl. Med. 2014, 12, 241.-   11. Powell Jr, D. J., Brennan, A. L., Zheng, Z., et al. Efficient    Clinical-scale Enrichment of Lymphocytes for Use in Adoptive    Immunotherapy Using a Modified Counterflow Centrifugal Elutriation    Program. Cytotherapy 2009, 11, 923-935.-   12. TerumoBCT. ELUTRA Cell Separation System. Manufacturer    recommendations for the Enrichment of Lymphocytes from Apheresis    Residues.-   13. C. E. Chiche-Lapierre, C. E., Tramalloni, D, Chaput, N. et al.    Comparative Analysis of Sepax S-100, COBE 2991, and Manual DMSO    Removal Techniques From Cryopreserved Hematopoietic Stem Cell    Apheresis Product Cytotherapy 2016 18, 6:S47.-   14. Huang, L, Cox, E, Austin, R. Continuous particle separation    through deterministic lateral displacement, Science 2004,    304:987-990.-   15. Davis, J. A., Inglis, D. W., et al. Deterministic Hydrodynamics:    Taking Blood Apart. Proc. Natl. Acad. Sci. USA 2006, 103,    14779-14784.-   16. Inglis, D. W., Davis, J. A., Austin, R. H. Critical particle    Size for Fractionation by Deterministic Lateral Displacement. Lab    Chip 2006, 6, 655-658. 17. Chen, Y, D'Silva, J. Austin, R et al.    Microfluidic chemical processing with on-chip washing by    deterministic lateral displacement arrays with separator walls.    Biomicrofluidics. 2015 9(5): 054105.-   18. Shilun, F. Skelley, A., Anwer, A. G., et al. Maximizing Particle    Concentration in Deterministic Lateral Displacement Arrays.    Biomicrofluidics 2017, 11, 024121.-   19. D'Silva, J. Throughout Microfluidic Capture of Rare Cells from    Large Volumes of Blood. A Dissertation Presented to the Faculty of    Princeton University in Candidacy for the Degree of Doctor of    Philosophy. 2016.-   20. NIST/SEMATECH e-Handbook of Statistical Methods, 2017,    www.itl.nist.gov/div898/handbook/August-   21. Mahnke, Y. D., Brodie, T. M., Sallusto, F., et al. The Who's Who    of T-cell Differentiation: Human Memory T-cell Subsets. Eur. J. of    Immunol. 2013, 43, 2797-2809.-   22. Civin, C. I., Ward, T., Skelley, A. M., et al. Automated    Leukocyte Processing by Microfluidic Deterministic Lateral    Displacement. Cytometry A. 2016, 89, 1073-1083.-   23. Trickett, A., and Kwan, Y. L. T-cell Stimulation and Expansion    Using Anti-CD3/CD28 Beads. J. Immunol. Meth. 2003, 275, 251-255.-   24. Marktkamcham, S., Onlamoon, N., Wang, S., et al. The Effects of    Anti-CD3/CD28 Coated Beads and IL-2 on Expanded T-cell for    Immunotherapy. Adv. Clin. Exp. Med. 2016, 25, 821-828.-   25. Li, Y., and Kurlander, R. J. Comparison of Anti-CD3 and    Anti-CD28-coated Beads with Soluble Anti-CD3 for Expanding Human    T-cells: Differing Impact on CD8 T-cell Phenotype and    Responsiveness. J. Transl. Med. 2010, 8, 104-118.-   26. Agrawal S., Ganguly, S., Hjian, P., et al. PDGF Upregulates    CLEC-2 to Induce T Regulatory Cells. Oncotarget. 2015, 6,    28621-28632.-   27. Zhu, L., Huang, Z., Stålesen, R. et al. Platelets Provoke    Distinct Dynamics of Immune Response by Differentially Regulating    CD4+ T-cell Proliferation. J. Throm. Haem. 2014, 12, 1156-1165.-   28. Koesdjojo, M., Lee, Z., Dosier, C., et al. DLD Microfluidic    Purification and Characterization of Intact and Viable Circulating    Tumor Cells in Peripheral Blood. AACR Annual Meeting 2016, Abstract    #3956.-   29. Loutherback, K. “Microfluidic Devices for High Throughput Cell    Sorting and Chemical Treatment,” A Dissertation Presented to the    Faculty of Princeton University 2011.

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by one of skillin the art that the invention may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodiment thereof

What is claimed is:
 1. A method for preparing central memory T cellsfrom an apheresis or leukapheresis sample, comprising: a) purifying thecentral memory T cells from the sample by a combination of aDeterministic Lateral Displacement (DLD) step, and in addition to theDLD step, an affinity separation step; and b) expanding the centralmemory T cells purified in step a) by culturing the central memory Tcells in the presence of an activator.
 2. The method of claim 1, whereinafter the central memory T cells have been purified by DLD and saidaffinity step, they are genetically engineered to express atherapeutically active protein.
 3. The method of claim 1, wherein,before DLD is performed, the central memory T cells are bound to one ormore carriers in a way that promotes DLD separation.
 4. The method ofclaim 3, wherein said one or more carriers comprise an antibody oractivator that binds specifically to T cells.
 5. The method of claim 4,wherein said one or more carriers are magnetized.
 6. The method of claim4, wherein the carriers comprise an anti CD3 antibody.
 7. The method ofclaim 1, wherein during the expansion of cells by culturing in thepresence of activator, a greater percentage of cells express cluster ofdifferentiation (CD) antigens characteristic of a memory phenotype thancells obtained by affinity separation alone.
 8. The method of claim 1,wherein said apheresis or leukapheresis sample is obtained from apatient with cancer, an autoimmune disease or an infectious disease. 9.The method of claim 1 wherein DLD is performed on a microfluidic devicecomprising: a) at least one channel extending from a sample inlet to oneor more fluid outlets, wherein the channel is bounded by a first walland a second wall opposite from the first wall; b) an array of obstaclesarranged in rows in the channel, each subsequent row of obstacles beingshifted laterally with respect to a previous row, and wherein saidobstacles are disposed in a manner such that, when the apheresis orleukapheresis sample is applied to an inlet of the device andfluidically passed through the channel, T cells in the composition flowto one or more collection outlets where an enriched product iscollected, and cells, or particles that are in the apheresis orleukapheresis sample and that are of a different size than the T cells,flow to one more waste outlets that are separate from the collectionoutlets.
 10. The method of claim 9, wherein the affinity separation stepcomprises binding the T cells to magnetic beads that bind specificallyto T cells.
 11. The method of claim 10, wherein said DLD is performedbefore said affinity separation step.
 12. The method of claim 1, whereinthe affinity separation step comprises binding the central memory Tcells to magnetic beads that bind specifically to T cells.
 13. Themethod of claim 1, wherein after the central memory T cells have beenpurified by said DLD and said affinity step, they are geneticallyengineered to produce chimeric antigen receptors (CARs) on theirsurface.
 14. The method of claim 13, wherein the apheresis sample isobtained from a patient with cancer and the genetically engineered cellsare administered to the same patient.
 15. The method of claim 14,wherein the yield of T cells expressing the chimeric receptors on theirsurface is at least 20% greater than T cells isolated by apheresis orleukapheresis and subjected to magnetic separation but not DLD.
 16. Themethod of claim 13, wherein DLD is performed on a microfluidic devicecomprising: a) at least one channel extending from a sample inlet to oneor more fluid outlets, wherein the channel is bounded by a first walland a second wall opposite from the first wall; b) an array of obstaclesarranged in rows in the channel, each subsequent row of obstacles beingshifted laterally with respect to a previous row, and wherein saidobstacles are disposed in a manner such that, when the apheresis orleukapheresis sample is applied to an inlet of the device andfluidically passed through the channel, T cells in the composition flowto one or more collection outlets where an enriched product iscollected, and cells, or particles that are in the apheresis orleukapheresis sample and that are of a different size than the T cells,flow to one more waste outlets that are separate from the collectionoutlets.
 17. The method of claim 16, wherein the affinity separationstep comprises binding the central memory T cells to magnetic beads thatbind specifically to T cells.
 18. The method of claim 17, wherein saidDLD is performed before said affinity separation step.
 19. The method ofclaim 13, wherein the yield of central memory T cells expressingchimeric receptors on their surface after purification, geneticengineering and expansion is at least 10% greater than central memory Tcells prepared in the same manner but not subjected to DLD.
 20. Themethod of claim 13, wherein the affinity separation step comprisesbinding the central memory T cells to magnetic beads that bindspecifically to T cells.